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Sample records for caspase cleavage ofmajor

  1. Nuclear substructure reorganization during late stageerythropoiesis is selective and does not involve caspase cleavage ofmajor nuclear substructural proteins

    SciTech Connect

    Krauss, Sharon Wald; Lo, Annie J.; Short, Sarah A.; Koury, MarkJ.; Mohandas, Narla; Chasis, Joel Anne

    2005-04-06

    Enucleation, a rare feature of mammalian differentiation, occurs in three cell types: erythroblasts, lens epithelium and keratinocytes. Previous investigations suggest that caspase activation functions in lens epithelial and keratinocyte enucleation, as well as in early erythropoiesis encompassing BFU-E differentiation to proerythroblast. To determine whether caspase activation contributes to later erythropoiesis and whether nuclear substructures other than chromatin reorganize, we analyzed distributions of nuclear subcompartment proteins and assayed for caspase-induced cleavage of subcompartmental target proteins in mouse erythroblasts. We found that patterns of lamin B in the filamentous network interacting with both the nuclear envelope and DNA, nuclear matrix protein NuMA, and splicing factors Sm and SC35 persisted during nuclear condensation, consistent with effective transcription of genes expressed late in differentiation. Thus nuclear reorganization prior to enucleation is selective, allowing maintenance of critical transcriptional processes independent of extensive chromosomal reorganization. Consistent with these data, we found no evidence for caspase-induced cleavage of major nuclear subcompartment proteins during late erythropoiesis, in contrast to what has been observed in early erythropoiesis and in lens epithelial and keratinocyte differentiation. These findings imply that nuclear condensation and extrusion during terminal erythroid differentiation involve novel mechanisms that do not entail major activation of apoptotic machinery.

  2. Limited caspase cleavage of human BAP31.

    PubMed

    Määttä, J; Hallikas, O; Welti, S; Hildén, P; Schröder, J; Kuismanen, E

    2000-11-10

    Human BAP31 was cleaved at both of its two identical caspase cleavage sites in two previously reported models of apoptosis. We show here that only the most carboxy-terminal site is cleaved during apoptosis induced in HeLa cells by tunicamycin, tumor necrosis factor and cycloheximide, or staurosporine. Similar results were obtained in HL-60 cells using Fas/APO-1 antibodies, or cycloheximide. This limited cleavage, which is inhibited by several caspase inhibitors, removes eight amino acids from human BAP31 including the KKXX coat protein I binding motif. Ectopic expression of the resulting cleavage product induces redistribution of mannosidase II from the Golgi and prevents endoplasmic reticulum to Golgi transport of virus glycoproteins.

  3. Interactome disassembly during apoptosis occurs independent of caspase cleavage.

    PubMed

    Scott, Nichollas E; Rogers, Lindsay D; Prudova, Anna; Brown, Nat F; Fortelny, Nikolaus; Overall, Christopher M; Foster, Leonard J

    2017-01-12

    Protein-protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas-mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome-wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.

  4. Caspase-2 cleavage of tau reversibly impairs memory.

    PubMed

    Zhao, Xiaohui; Kotilinek, Linda A; Smith, Benjamin; Hlynialuk, Chris; Zahs, Kathleen; Ramsden, Martin; Cleary, James; Ashe, Karen H

    2016-11-01

    In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

  5. Caspase-mediated specific cleavage of human histone deacetylase 4.

    PubMed

    Liu, Fang; Dowling, Melissa; Yang, Xiang-Jiao; Kao, Gary D

    2004-08-13

    Histone deacetylase 4 (HDAC4) is a class II HDAC implicated in controlling gene expression important for diverse cellular functions, but little is known about how its expression and stability are regulated. We report here that this deacetylase is unusually unstable, with a half-life of less than 8 h. Consistent with the instability of HDAC4 protein, its mRNA was also highly unstable (with a half-life of less than 4 h). The degradation of HDAC4 could be accelerated by exposure of cells to ultraviolet irradiation. HDAC4 degradation was not dependent on proteasome or CRM1-mediated export activity but instead was caspase-dependent and was detectable in diverse human cancer lines. Of two potential caspase consensus motifs in HDAC4, both lying within a region containing proline-, glutamic acid-, serine-, and threonine-rich (PEST) sequences, we identified, by site-directed mutagenesis, Asp-289 as the prime cleavage site. Notably, this residue is not conserved among other class IIa members, HDAC5, -7, and -9. Finally, the induced expression of caspase-cleavable HDAC4 led to markedly increased apoptosis. These results therefore unexpectedly link the regulation of HDAC4 protein stability to caspases, enzymes that are important for controlling cell death and differentiation.

  6. Predicting caspase substrate cleavage sites based on a hybrid SVM-PSSM method.

    PubMed

    Li, Dandan; Jiang, Zhenran; Yu, Weiming; Du, Lei

    2010-12-01

    Caspases play an important role in many critical non-apoptosis processes by cleaving relevant substrates at cleavage sites. Identification of caspase substrate cleavage sites is the key to understand these processes. This paper proposes a hybrid method using support vector machine (SVM) in conjunction with position specific scoring matrices (PSSM) for caspase substrate cleavage sites prediction. Three encoding schemes including orthonormal binary encoding, BLOSUM62 matrix profile and PSSM profile of neighborhood surrounding the substrate cleavage sites were regarded as the input of SVM. The 10-fold cross validation results demonstrate that the SVM-PSSM method performs well with an overall accuracy of 97.619% on a larger dataset.

  7. Proteinase 3-dependent caspase-3 cleavage modulates neutrophil death and inflammation.

    PubMed

    Loison, Fabien; Zhu, Haiyan; Karatepe, Kutay; Kasorn, Anongnard; Liu, Peng; Ye, Keqiang; Zhou, Jiaxi; Cao, Shannan; Gong, Haiyan; Jenne, Dieter E; Remold-O'Donnell, Eileen; Xu, Yuanfu; Luo, Hongbo R

    2014-10-01

    Caspase-3-mediated spontaneous death in neutrophils is a prototype of programmed cell death and is critical for modulating physiopathological inflammatory responses; however, the underlying regulatory pathways remain ill defined. Here we determined that in aging neutrophils, the cleavage and activation of caspase-3 is independent of the canonical caspase-8- or caspase-9-mediated pathway. Instead, caspase-3 activation was mediated by serine protease proteinase 3 (PR3), which is present in the cytosol of aging neutrophils. Specifically, PR3 cleaved procaspase-3 at a site upstream of the canonical caspase-9 cleavage site. In mature neutrophils, PR3 was sequestered in granules and released during aging via lysosomal membrane permeabilization (LMP), leading to procaspase-3 cleavage and apoptosis. Pharmacological inhibition or knockdown of PR3 delayed neutrophil death in vitro and consistently delayed neutrophil death and augmented neutrophil accumulation at sites of inflammation in a murine model of peritonitis. Adoptive transfer of both WT and PR3-deficient neutrophils revealed that the delayed death of neutrophils lacking PR3 is due to an altered intrinsic apoptosis/survival pathway, rather than the inflammatory microenvironment. The presence of the suicide protease inhibitor SERPINB1 counterbalanced the protease activity of PR3 in aging neutrophils, and deletion of Serpinb1 accelerated neutrophil death. Taken together, our results reveal that PR3-mediated caspase-3 activation controls neutrophil spontaneous death.

  8. Proteinase 3–dependent caspase-3 cleavage modulates neutrophil death and inflammation

    PubMed Central

    Loison, Fabien; Zhu, Haiyan; Karatepe, Kutay; Kasorn, Anongnard; Liu, Peng; Ye, Keqiang; Zhou, Jiaxi; Cao, Shannan; Gong, Haiyan; Jenne, Dieter E.; Remold-O’Donnell, Eileen; Xu, Yuanfu; Luo, Hongbo R.

    2014-01-01

    Caspase-3–mediated spontaneous death in neutrophils is a prototype of programmed cell death and is critical for modulating physiopathological inflammatory responses; however, the underlying regulatory pathways remain ill defined. Here we determined that in aging neutrophils, the cleavage and activation of caspase-3 is independent of the canonical caspase-8– or caspase-9–mediated pathway. Instead, caspase-3 activation was mediated by serine protease proteinase 3 (PR3), which is present in the cytosol of aging neutrophils. Specifically, PR3 cleaved procaspase-3 at a site upstream of the canonical caspase-9 cleavage site. In mature neutrophils, PR3 was sequestered in granules and released during aging via lysosomal membrane permeabilization (LMP), leading to procaspase-3 cleavage and apoptosis. Pharmacological inhibition or knockdown of PR3 delayed neutrophil death in vitro and consistently delayed neutrophil death and augmented neutrophil accumulation at sites of inflammation in a murine model of peritonitis. Adoptive transfer of both WT and PR3-deficient neutrophils revealed that the delayed death of neutrophils lacking PR3 is due to an altered intrinsic apoptosis/survival pathway, rather than the inflammatory microenvironment. The presence of the suicide protease inhibitor SERPINB1 counterbalanced the protease activity of PR3 in aging neutrophils, and deletion of Serpinb1 accelerated neutrophil death. Taken together, our results reveal that PR3-mediated caspase-3 activation controls neutrophil spontaneous death. PMID:25180606

  9. Cascleave 2.0, a new approach for predicting caspase and granzyme cleavage targets.

    PubMed

    Wang, Mingjun; Zhao, Xing-Ming; Tan, Hao; Akutsu, Tatsuya; Whisstock, James C; Song, Jiangning

    2014-01-01

    Caspases and granzyme B (GrB) are important proteases involved in fundamental cellular processes and play essential roles in programmed cell death, necrosis and inflammation. Although a number of substrates for both types have been experimentally identified, the complete repertoire of caspases and granzyme B substrates remained to be fully characterized. Accordingly, systematic bioinformatics studies of known cleavage sites may provide important insights into their substrate specificity and facilitate the discovery of novel substrates. We develop a new bioinformatics tool, termed Cascleave 2.0, which builds on previous success of the Cascleave tool for predicting generic caspase cleavage sites. It can be efficiently used to predict potential caspase-specific cleavage sites for the human caspase-1, 3, 6, 7, 8 and GrB. In particular, we integrate heterogeneous sequence and protein functional information from various sources to improve the prediction accuracy of Cascleave 2.0. During classification, we use both maximum relevance minimum redundancy and forward feature selection techniques to quantify the relative contribution of each feature to prediction and thus remove redundant as well as irrelevant features. A systematic evaluation of Cascleave 2.0 using the benchmark data and comparison with other state-of-the-art tools using independent test data indicate that Cascleave 2.0 outperforms other tools on protease-specific cleavage site prediction of caspase-1, 3, 6, 7 and GrB. Cascleave 2.0 is anticipated to be used as a powerful tool for identifying novel substrates and cleavage sites of caspases and GrB and help understand the functional roles of these important proteases in human proteolytic cascades. http://www.structbioinfor.org/cascleave2/.

  10. Appoptosin-Mediated Caspase Cleavage of Tau Contributes to Progressive Supranuclear Palsy Pathogenesis.

    PubMed

    Zhao, Yingjun; Tseng, I-Chu; Heyser, Charles J; Rockenstein, Edward; Mante, Michael; Adame, Anthony; Zheng, Qiuyang; Huang, Timothy; Wang, Xin; Arslan, Pharhad E; Chakrabarty, Paramita; Wu, Chengbiao; Bu, Guojun; Mobley, William C; Zhang, Yun-Wu; St George-Hyslop, Peter; Masliah, Eliezer; Fraser, Paul; Xu, Huaxi

    2015-09-02

    Progressive supranuclear palsy (PSP) is a movement disorder characterized by tau neuropathology where the underlying mechanism is unknown. An SNP (rs1768208 C/T) has been identified as a strong risk factor for PSP. Here, we identified a much higher T-allele occurrence and increased levels of the pro-apoptotic protein appoptosin in PSP patients. Elevations in appoptosin correlate with activated caspase-3 and caspase-cleaved tau levels. Appoptosin overexpression increased caspase-mediated tau cleavage, tau aggregation, and synaptic dysfunction, whereas appoptosin deficiency reduced tau cleavage and aggregation. Appoptosin transduction impaired multiple motor functions and exacerbated neuropathology in tau-transgenic mice in a manner dependent on caspase-3 and tau. Increased appoptosin and caspase-3-cleaved tau were also observed in brain samples of patients with Alzheimer's disease and frontotemporal dementia with tau inclusions. Our findings reveal a novel role for appoptosin in neurological disorders with tau neuropathology, linking caspase-3-mediated tau cleavage to synaptic dysfunction and behavioral/motor defects.

  11. Role for Caspase-Mediated Cleavage of Rad51 in Induction of Apoptosis by DNA Damage

    PubMed Central

    Huang, YinYin; Nakada, Shuji; Ishiko, Takatoshi; Utsugisawa, Taiju; Datta, Rakesh; Kharbanda, Surender; Yoshida, Kiyotsugu; Talanian, Robert V.; Weichselbaum, Ralph; Kufe, Donald; Yuan, Zhi-Min

    1999-01-01

    We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage. PMID:10082566

  12. Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8.

    PubMed

    Desagher, S; Osen-Sand, A; Montessuit, S; Magnenat, E; Vilbois, F; Hochmann, A; Journot, L; Antonsson, B; Martinou, J C

    2001-09-01

    Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.

  13. RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1

    PubMed Central

    Siddiqui, Mohammad Adnan; Mukherjee, Sushovita; Manivannan, Praveen; Malathi, Krishnamurthy

    2015-01-01

    Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer. PMID:26263979

  14. Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release.

    PubMed

    Zhu, Yushan; Li, Min; Wang, Xiaohui; Jin, Haijing; Liu, Shusen; Xu, Jianxin; Chen, Quan

    2012-01-01

    Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions. The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood. Here we addressed the underlying mechanism, explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome c (cyto.c) release. We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3). We found that cyto.c1 was cleaved at the site of D106, which is critical for binding with cyto.c, following apoptotic stresses or targeted expression of casp.3 into the mitochondrial intermembrane space. We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe. These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk. Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.

  15. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

    PubMed Central

    Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-01-01

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

  16. HIV-1 Vpu Accessory Protein Induces Caspase-mediated Cleavage of IRF3 Transcription Factor*

    PubMed Central

    Park, Sang Yoon; Waheed, Abdul A.; Zhang, Zai-Rong; Freed, Eric O.; Bonifacino, Juan S.

    2014-01-01

    Vpu is an accessory protein encoded by HIV-1 that interferes with multiple host-cell functions. Herein we report that expression of Vpu by transfection into 293T cells causes partial proteolytic cleavage of interferon regulatory factor 3 (IRF3), a key transcription factor in the innate anti-viral response. Vpu-induced IRF3 cleavage is mediated by caspases and occurs mainly at Asp-121. Cleavage produces a C-terminal fragment of ∼37 kDa that comprises the IRF dimerization and transactivation domains but lacks the DNA-binding domain. A similar cleavage is observed upon infection of the Jurkat T-cell line with vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1. Two other HIV-1 accessory proteins, Vif and Vpr, also contribute to the induction of IRF3 cleavage in both the transfection and the infection systems. The C-terminal IRF3 fragment interferes with the transcriptional activity of full-length IRF3. Cleavage of IRF3 under all of these conditions correlates with cleavage of poly(ADP-ribose) polymerase, an indicator of apoptosis. We conclude that Vpu contributes to the attenuation of the anti-viral response by partial inactivation of IRF3 while host cells undergo apoptosis. PMID:25352594

  17. Human iPSC-MSC-Derived Xenografts Modulate Immune Responses by Inhibiting the Cleavage of Caspases.

    PubMed

    Li, Cheng-Lin; Leng, Yun; Zhao, Bin; Gao, Chang; Du, Fei-Fei; Jin, Ning; Lian, Qi-Zhou; Xu, Shuang-Yue; Yan, Guo-Liang; Xia, Jun-Jie; Zhuang, Guo-Hong; Fu, Qing-Ling; Qi, Zhong-Quan

    2017-07-01

    Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-β1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732. © 2017 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  18. CaspNeuroD: a knowledgebase of predicted caspase cleavage sites in human proteins related to neurodegenerative diseases

    PubMed Central

    Kumar, Sonu; Cieplak, Piotr

    2016-01-01

    Background: A variety of neurodegenerative diseases (NDs) have been associated with deregulated caspase activation that leads to neuronal death. Caspases appear to be involved in the molecular pathology of NDs by directly cleaving important proteins. For instance, several proteins involved in Alzheimer’s disease, including β-amyloid precursor protein (APP) and presenilins, are known to be cleaved by caspases. Therefore, cell death pathway may play a central role in many neurological diseases, and targeting the important proteins that control the cell survival and death may potentially represent a therapeutic approach for chronic neurodegenerative disorders. Findings: We developed CaspNeuroD, a relational database of in silico predicted caspase cleavage sites in human proteins associated with NDs. The prediction has been done on collection of 249 human proteins reported in clinical studies of NDs using the recently published CaspDB Random Forest machine-learning model. This database could be used for identifying new caspase substrates and further our understanding of the caspase-mediated substrate cleavage in NDs. Conclusion: Our database provides information about potential caspase cleavage sites in a verified set of human proteins involved in NDs. It provides also information about the conservation of cleavage positions in corresponding orthologs, and information about the positions of single nucleotide polymorphisms and posttranslational modifications (PTMs) that may modulate the caspase cleavage efficiency. Database URL: caspdb.sanfordburnham.org/caspneurod.php . PMID:28025335

  19. Hypoxia-induced cleavage of caspase-3 and DFF45/ICAD in human failed cardiomyocytes.

    PubMed

    Todor, Anastassia; Sharov, Victor G; Tanhehco, Elaine J; Silverman, Norman; Bernabei, Alvise; Sabbah, Hani N

    2002-09-01

    It has been proposed that the hemodynamic deterioration associated with heart failure (HF) may be due in part to ongoing loss of viable cardiac myocytes through apoptosis. Hypoxia has been shown to promote apoptosis in normal cardiomyocytes. Adaptation and maladaptations inherent to heart failure can modify the susceptibility of cells to different stress factors. We hypothesized that HF modifies the threshold of cardiomyocytes to hypoxia-induced apoptosis. Cardiomyocytes were isolated from 18 human hearts explanted at the time of cardiac transplantation due to either ischemic cardiomyopathy (ICM) (n = 9) or idiopathic dilated cardiomyopathy (IDC) (n = 9). Tissue from five normal donor hearts (NL) for whom no suitable recipient was available was used as control. Cardiomyocytes were incubated for 3 h under normoxic (95% air-5% CO(2)) or hypoxic (95% N(2)-5% CO(2)) conditions. Expression of caspase-3 and DNA fragmentation factor-45 (DFF45)/inhibitor of caspase-3-activated DNase (ICAD) was detected by Western blot analysis. Three hours of hypoxia did not affect the expression of these proteins in NL cardiomyocytes. In contrast, hypoxia led to cleavage of caspase-3 and DFF45/ICAD both in ICM and IDC. In conclusion, failing cardiomyocytes exhibit increased susceptibility to hypoxia-induced apoptosis.

  20. Cleavage of the transactivation-inhibitory domain of p63 by caspases enhances apoptosis.

    PubMed

    Sayan, Berna S; Sayan, A Emre; Yang, Ai Li; Aqeilan, Rami I; Candi, Eleonora; Cohen, Gerald M; Knight, Richard A; Croce, Carlo M; Melino, Gerry

    2007-06-26

    p63 is a p53-related transcription factor. Utilization of two different promoters and alternative splicing at the C terminus lead to generation of six isoforms. The alpha isoforms of TAp63 and DeltaNp63 contain a transactivation-inhibitory (TI) domain at the C termini, which can bind to the transactivation (TA) domain and inhibit its transcriptional activity. Consequently, TAp63alpha can directly inhibit its activity through an intramolecular interaction; similarly, DeltaNp63alpha can inhibit the activity of the active TAp63 isoforms through an intermolecular interaction. In this work, we demonstrate that after induction of apoptosis, the TI domain of the p63alpha isoforms is cleaved by activated caspases. Cleavage of DeltaNp63alpha relieves its inhibitory effect on the transcriptionally active p63 proteins, and the cleavage of TAp63alpha results in production of a TAp63 protein with enhanced transcriptional activity. In agreement with these data, generation of the N-terminal TAp63 fragment has a role in apoptosis because stable cell lines expressing wild-type TAp63 are more sensitive to apoptosis compared with cells expressing the noncleavable mutant. We also used a model system in which TAp63 expression was induced by trichostatin-A treatment in HCT116 cells. Trichostatin-A sensitized these cells to apoptosis, and this sensitization was associated with cleavage of up-regulated p63.

  1. Initiation of Apoptosis by Granzyme B Requires Direct Cleavage of Bid, but Not Direct Granzyme B–Mediated Caspase Activation

    PubMed Central

    Sutton, Vivien R.; Davis, Joanne E.; Cancilla, Michael; Johnstone, Ricky W.; Ruefli, Astrid A.; Sedelies, Karin; Browne, Kylie A.; Trapani, Joseph A.

    2000-01-01

    The essential upstream steps in granzyme B–mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B–resistant Bcl-2–overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B–treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B. PMID:11085743

  2. Scarless fetal mouse wound healing may initiate apoptosis through caspase 7 and cleavage of PARP.

    PubMed

    Carter, Richard; Sykes, Virginia; Lanning, David

    2009-09-01

    Apoptotic mechanisms are thought to be important in wound healing for the removal of inflammatory cells and evolution of granulation tissue. However, little is understood about the signal, propagation, and mechanisms responsible for triggering cell death in tissue injury, particularly during fetal wound repair. Understanding these signals may lead to insights regarding scarless wound healing. We hypothesized that differences in apoptosis would exist in mid- (E15) compared with late-gestational (E18) mice subjected to cutaneous wounds. We examined early apoptotic signals that may be initiated following tissue injury. Pregnant, time-dated mice underwent laparotomy and hysterotomy on embryonic day 15 (E15) and 18 (E18). Full-thickness, excisional cutaneous wounds were made on the dorsum of the fetuses and dorsal skin harvested 15 and 45 min after wounding. Unwounded dorsal skin from additional fetuses collected at the same time points served as controls. The skin was processed to obtain protein, then levels of caspase 3, caspase 7, and poly ADP-ribose polymerase (PARP) were measured by Western blot. Cyclophilin levels were measured to ensure equal loading of protein. Histone-associated DNA complex formation was examined to provide further evidence of cellular apoptosis. There were no differences in total caspase 3 levels between E15 and E18 fetal tissue with or without wounding, nor was any cleavage of caspase 3 noted in any group. However, cleaved caspase 7 was present in the E15 skin with a >2-fold increase following wounding at both 15 and 45 min, yet absent in the E18 groups. Furthermore, levels of cleaved PARP were also increased by >2-fold at both 15 and 45 min in E15 wound groups, whereas a relatively small amount was only seen in the E18 wound groups at 45 min. DNA-histone fragmentation ELISA assay showed a 5-fold increase in the enrichment of histone-associated DNA fragments in the E15 wounded tissue compared with the time-matched controls at 45 min. This was

  3. The caspase-generated cleavage product of Ets-1 p51 and Ets-1 p27, Cp17, induces apoptosis.

    PubMed

    Choul-Li, Souhaila; Tulasne, David; Aumercier, Marc

    2016-11-04

    The transcription factor Ets-1 is involved in various physiological processes and invasive pathologies. Human Ets-1 exists under three isoforms: p51, the predominant full-length isoform, p42 and p27, shorter alternatively spliced isoforms. We have previously demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed by caspases in vitro and during apoptosis. However, the caspase cleavage of the second spliced variant Ets-1 p27 remains to investigate. In the present study, we demonstrate that Ets-1 p27 is a cleavage substrate of caspases. We show that Ets-1 p27 is processed in vitro by caspase-3, resulting in three C-terminal fragments Cp20, Cp17 and Cp14. Similarly, Ets-1 p27 was cleaved during apoptotic cell death induced by anisomycin, producing fragments consistent with those observed in in vitro cleavage assay. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p27. As a functional consequences, Cp17 fragment, the major cleavage product generated during apoptosis, induced itself apoptosis when transfected into cells. Our results show that Ets-1 p27 is cleaved in the same manner as Ets-1 p51 within the exon VII-encoded region, thus generating a stable C-terminal fragment that induces cell death by initiating apoptosis.

  4. Caspase-mediated cleavage of C53/LZAP protein causes abnormal microtubule bundling and rupture of the nuclear envelope.

    PubMed

    Wu, Jianchun; Jiang, Hai; Luo, Shouqing; Zhang, Mingsheng; Zhang, Yinghua; Sun, Fei; Huang, Shuang; Li, Honglin

    2013-05-01

    Apoptotic nucleus undergoes distinct morphological and biochemical changes including nuclear shrinkage, chromatin condensation and DNA fragmentation, which are attributed to caspase-mediated cleavage of several nuclear substrates such as lamins. As most of active caspases reside in the cytoplasm, disruption of the nuclear-cytoplasmic barrier is essential for caspases to reach their nuclear targets. The prevailing proposed mechanism is that the increase in the permeability of nuclear pores induced by caspases allows the caspases and other apoptotic factors to diffuse into the nucleus, thereby resulting in the nuclear destruction. Here, we report a novel observation that physical rupture of the nuclear envelope (NE) occurs in the early stage of apoptosis. We found that the NE rupture was caused by caspase-mediated cleavage of C53/LZAP, a protein that has been implicated in various signaling pathways, including NF-κB signaling and DNA damage response, as well as tumorigenesis and metastasis. We also demonstrated that C53/LZAP bound indirectly to the microtubule (MT), and expression of the C53/LZAP cleavage product caused abnormal MT bundling and NE rupture. Taken together, our findings suggest a novel role of C53/LZAP in the regulation of MT dynamics and NE structure during apoptotic cell death. Our study may provide an additional mechanism for disruption of the nuclear-cytoplasmic barrier during apoptosis.

  5. Caspase-mediated cleavage of C53/LZAP protein causes abnormal microtubule bundling and rupture of the nuclear envelope

    PubMed Central

    Wu, Jianchun; Jiang, Hai; Luo, Shouqing; Zhang, Mingsheng; Zhang, Yinghua; Sun, Fei; Huang, Shuang; Li, Honglin

    2013-01-01

    Apoptotic nucleus undergoes distinct morphological and biochemical changes including nuclear shrinkage, chromatin condensation and DNA fragmentation, which are attributed to caspase-mediated cleavage of several nuclear substrates such as lamins. As most of active caspases reside in the cytoplasm, disruption of the nuclear-cytoplasmic barrier is essential for caspases to reach their nuclear targets. The prevailing proposed mechanism is that the increase in the permeability of nuclear pores induced by caspases allows the caspases and other apoptotic factors to diffuse into the nucleus, thereby resulting in the nuclear destruction. Here, we report a novel observation that physical rupture of the nuclear envelope (NE) occurs in the early stage of apoptosis. We found that the NE rupture was caused by caspase-mediated cleavage of C53/LZAP, a protein that has been implicated in various signaling pathways, including NF-κB signaling and DNA damage response, as well as tumorigenesis and metastasis. We also demonstrated that C53/LZAP bound indirectly to the microtubule (MT), and expression of the C53/LZAP cleavage product caused abnormal MT bundling and NE rupture. Taken together, our findings suggest a novel role of C53/LZAP in the regulation of MT dynamics and NE structure during apoptotic cell death. Our study may provide an additional mechanism for disruption of the nuclear-cytoplasmic barrier during apoptosis. PMID:23478299

  6. Lens fiber connexin turnover and caspase-3-mediated cleavage are regulated alternately by phosphorylation.

    PubMed

    Yin, Xinye; Liu, Jialu; Jiang, Jean X

    2008-05-01

    Lens connexins are phosphorylated in vivo; however, the function and regulation of the phosphorylation remain largely unknown. We have previously identified an in vivo phosphorylation site, Ser(364), at the COOH terminus of lens connexin (Cx) Cx45.6 and phosphorylation appears to regulate connexin protein turnover. To assess the specific mechanism of Ser(364) phosphorylation in Cx45.6, exogenous wild type and Ser(364) mutant Cx45.6 were expressed in primary lens cultures through retroviral infection. Cx45.6 turnover was attenuated primarily by proteasomal inhibitors and to a lesser extent by lysosomal inhibitors. Furthermore, the level of Cx45.6 protein in ubiquitin co-expressed cells was significantly reduced as compared to the cells expressing Cx45.6 alone. Moreover, overexpression of ubiquitin led to a more significant decrease in wild type Cx45.6 than Cx45.6(S364A), a mutant deficient of phosphorylation site at Ser(364), although we did not detect any difference in the levels of ubiquitination between wild type and mutant Cx45.6. Interestingly, the mutant mimicking constitutive phosphorylation, Cx45.6(S364D), partially prevented the cleavage of Cx45.6 by caspase-3. Together, our data suggest that phosphorylation of Cx45.6 at Ser(364) appears to stimulate Cx45.6 turnover primarily through proteasome pathway and this phosphorylation inhibits the cleavage of Cx45.6 by caspase-3. These findings provide further insights into regulatory mechanism of the specific phosphorylation of connexins in the lens.

  7. Lens Fiber Connexin Turnover and Caspase-3-Mediated Cleavage Are Regulated Alternately by Phosphorylation

    PubMed Central

    Yin, Xinye; Liu, Jialu; Jiang, Jean X.

    2009-01-01

    Lens connexins are phosphorylated in vivo; however, the function and regulation of the phosphorylation remain largely unknown. We have previously identified an in vivo phosphorylation site, Ser364, at the COOH terminus of lens connexin (Cx) Cx45.6 and phosphorylation appears to regulate connexin protein turnover. To assess the specific mechanism of Ser364 phosphorylation in Cx45.6, exogenous wild type and Ser364 mutant Cx45.6 were expressed in primary lens cultures through retroviral infection. Cx45.6 turnover was attenuated primarily by proteasomal inhibitors and to a lesser extent by lysosomal inhibitors. Furthermore, the level of Cx45.6 protein in ubiquitin co-expressed cells was significantly reduced as compared to the cells expressing Cx45.6 alone. Moreover, overexpression of ubiquitin led to a more significant decrease in wild type Cx45.6 than Cx45.6(S364A), a mutant deficient of phosphorylation site at Ser364, although we did not detect any difference in the levels of ubiquitination between wild type and mutant Cx45.6. Interestingly, the mutant mimicking constitutive phosphorylation, Cx45.6(S364D), partially prevented the cleavage of Cx45.6 by caspase-3. Together, our data suggest that phosphorylation of Cx45.6 at Ser364appears to stimulate Cx45.6 turnover primarily through proteasome pathway and this phosphorylation inhibits the cleavage of Cx45.6 by caspase-3. These findings provide further insights into regulatory mechanism of the specific phosphorylation of connexins in the lens. PMID:18649174

  8. AKT1 induces caspase-mediated cleavage of the CDK inhibitor p27Kip1 during cell cycle progression in leukemia cells transformed by FLT3-ITD

    PubMed Central

    Yang, Xinping; Liu, Suiyang; Kharbanda, Surender; Stone, Richard M

    2011-01-01

    p27Kip1 cleavage and caspase-3 regulate cell cycle in human myeloma cells and B cells however regulation of p27Kip1 cleavage during the cell cycle is not known. In BaF3-FLT3-ITD cells, p27Kip1 undergoes C-terminal cleavage. Inhibition of the PI3K/AKT pathway is associated with decreased cleavage of p27Kip1 and G1 phase arrest. The caspase-3 inhibitor reduces p27Kip1 cleavage and inhibits cell proliferation. Knockdown shRNA against AKT1 reduces cleavage of p27Kip1, inhibits caspase-3 activation, and is associated with a delay in cell cycle progression. Taken together, these findings indicate that AKT1 induces caspase-mediated cleavage of p27Kip1, required for G1-S progression in FLT3-ITD cells. PMID:22142798

  9. Activation and cleavage of SASH1 by caspase-3 mediates an apoptotic response

    PubMed Central

    Burgess, Joshua T; Bolderson, Emma; Adams, Mark N; Baird, Anne-Marie; Zhang, Shu-Dong; Gately, Kathy A; Umezawa, Kazuo; O'Byrne, Kenneth J; Richard, Derek J

    2016-01-01

    Apoptosis is a highly regulated cellular process that functions to remove undesired cells from multicellular organisms. This pathway is often disrupted in cancer, providing tumours with a mechanism to avoid cell death and promote growth and survival. The putative tumour suppressor, SASH1 (SAM and SH3 domain containing protein 1), has been previously implicated in the regulation of apoptosis; however, the molecular role of SASH1 in this process is still unclear. In this study, we demonstrate that SASH1 is cleaved by caspase-3 following UVC-induced apoptosis. Proteolysis of SASH1 enables the C-terminal fragment to translocate from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild-type SASH1 or a cleaved form of SASH1 representing amino acids 231–1247 leads to an increase in apoptosis. Conversely, mutation of the SASH1 cleavage site inhibits nuclear translocation and prevents the initiation of apoptosis. SASH1 cleavage is also required for the efficient translocation of the transcription factor nuclear factor-κB (NF-κB) to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a codependence between SASH1 and NF-κB for this process. PMID:27831555

  10. Activation and cleavage of SASH1 by caspase-3 mediates an apoptotic response.

    PubMed

    Burgess, Joshua T; Bolderson, Emma; Adams, Mark N; Baird, Anne-Marie; Zhang, Shu-Dong; Gately, Kathy A; Umezawa, Kazuo; O'Byrne, Kenneth J; Richard, Derek J

    2016-11-10

    Apoptosis is a highly regulated cellular process that functions to remove undesired cells from multicellular organisms. This pathway is often disrupted in cancer, providing tumours with a mechanism to avoid cell death and promote growth and survival. The putative tumour suppressor, SASH1 (SAM and SH3 domain containing protein 1), has been previously implicated in the regulation of apoptosis; however, the molecular role of SASH1 in this process is still unclear. In this study, we demonstrate that SASH1 is cleaved by caspase-3 following UVC-induced apoptosis. Proteolysis of SASH1 enables the C-terminal fragment to translocate from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild-type SASH1 or a cleaved form of SASH1 representing amino acids 231-1247 leads to an increase in apoptosis. Conversely, mutation of the SASH1 cleavage site inhibits nuclear translocation and prevents the initiation of apoptosis. SASH1 cleavage is also required for the efficient translocation of the transcription factor nuclear factor-κB (NF-κB) to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a codependence between SASH1 and NF-κB for this process.

  11. The caspase-3 cleavage product of the plasma membrane Ca2+-ATPase 4b is activated and appropriately targeted.

    PubMed

    Pászty, Katalin; Antalffy, Géza; Penheiter, Alan R; Homolya, László; Padányi, Rita; Iliás, Attila; Filoteo, Adelaida G; Penniston, John T; Enyedi, Agnes

    2005-11-01

    The calmodulin-activated transporter hPMCA4 (human plasma membrane Ca2+-ATPase isoform 4) is a target for cleavage by caspase-3 during apoptosis. We have demonstrated that caspase-3 generates a 120 kDa fragment of this pump which lacks the complete autoinhibitory sequence [Paszty, Verma, Padanyi, Filoteo, Penniston and Enyedi (2002) J. Biol. Chem. 277, 6822-6829]. In the present study we analysed further the characteristics of the fragment of hPMCA4b produced by caspase-3. We did this by overexpressing the caspase-3 cleavage product of hPMCA4b in COS-7 and MDCKII (Madin-Darby canine kidney II) cells. This technique made it possible to clearly define the properties of this fragment, and we showed that it is constitutively active, as it forms a phosphoenzyme intermediate and has high Ca2+ transport activity in the absence of calmodulin. When this fragment of hPMCA4b was stably expressed in MDCKII cell clones, it was targeted without degradation to the basolateral plasma membrane. In summary, our studies emphasize that the caspase-3 cleavage product of hPMCA4b is constitutively active, and that the C-terminus is not required for proper targeting of hPMCA4b to the plasma membrane. Also, for the first time, we have generated cell clones that stably express a constitutively active PMCA.

  12. Caspase 3 - Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging

    PubMed Central

    Means, John C.; Gerdes, Bryan C.; Kaja, Simon; Sumien, Nathalie; Payne, Andrew J.; Stark, Danny A.; Borden, Priscilla K.; Price, Jeffrey L.; Koulen, Peter

    2016-01-01

    Mouse models of neurodegenerative diseases such as Alzheimer’s disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and in time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates to increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly negatively associated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau truncation and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain. PMID:27220334

  13. Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

    SciTech Connect

    Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan; Choe, Tae-Boo; Hong, Seok-Il; Lee, Yun-Han; Park, In-Chul

    2014-10-24

    Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

  14. p38 Activation Is Required Upstream of Potassium Current Enhancement and Caspase Cleavage in Thiol Oxidant-Induced Neuronal Apoptosis

    PubMed Central

    McLaughlin, BethAnn; Pal, Sumon; Tran, Minhnga P.; Parsons, Andrew A.; Barone, Frank C.; Erhardt, Joseph A.; Aizenman, Elias

    2013-01-01

    Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2′-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 μm DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDP-induced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death. PMID:11331359

  15. Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy.

    PubMed

    Granville, D J; Carthy, C M; Jiang, H; Shore, G C; McManus, B M; Hunt, D W

    1998-10-16

    Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other proapoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1-2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2-3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.

  16. Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death

    PubMed Central

    Martin, R; Desponds, C; Eren, R O; Quadroni, M; Thome, M; Fasel, N

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex regulating key pathways in cell growth. Hyperactivation of mTORC1 is implicated in numerous cancers, thus making it a potential broad-spectrum chemotherapeutic target. Here, we characterized how mTORC1 responds to cell death induced by various anticancer drugs such rapamycin, etoposide, cisplatin, curcumin, staurosporine and Fas ligand. All treatments induced cleavage in the mTORC1 component, raptor, resulting in decreased raptor–mTOR interaction and subsequent inhibition of the mTORC1-mediated phosphorylation of downstream substrates (S6K and 4E-BP1). The cleavage was primarily mediated by caspase-6 and occurred at two sites. Mutagenesis at one of these sites, conferred resistance to cell death, indicating that raptor cleavage is important in chemotherapeutic apoptosis. PMID:27551516

  17. Targeting of the transcription factor Max during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual glutamic acid residue in position P1.

    PubMed Central

    Krippner-Heidenreich, A; Talanian, R V; Sekul, R; Kraft, R; Thole, H; Ottleben, H; Lüscher, B

    2001-01-01

    Max is the central component of the Myc/Max/Mad network of transcription factors that regulate growth, differentiation and apoptosis. Whereas the Myc and Mad genes and proteins are highly regulated, Max expression is constitutive and no post-translational regulation is known. We have found that Max is targeted during Fas-induced apoptosis. Max is first dephosphorylated and subsequently cleaved by caspases. Two specific cleavage sites for caspases in Max were identified, one at IEVE(10) decreasing S and one at SAFD(135) decreasing G near the C-terminus, which are cleaved in vitro by caspase-5 and caspase-7 respectively. Mutational analysis indicates that both sites are also used in vivo. Thus Max represents the first caspase-5 substrate. The unusual cleavage after a glutamic acid residue is observed only with full-length, DNA-binding competent Max protein but not with corresponding peptides, suggesting that structural determinants might be important for this activity. Furthermore, cleavage by caspase-5 is inhibited by the protein kinase CK2-mediated phosphorylation of Max at Ser-11, a previously mapped phosphorylation site in vivo. These findings suggest that Fas-mediated dephosphorylation of Max is required for cleavage by caspase-5. The modifications that occur on Max in response to Fas signalling affect the DNA-binding activity of Max/Max homodimers. Taken together, our findings uncover three distinct processes, namely dephosphorylation and cleavage by caspase-5 and caspase-7, that target Max during Fas-mediated apoptosis, suggesting the regulation of the Myc/Max/Mad network through its central component. PMID:11535131

  18. GraBCas: a bioinformatics tool for score-based prediction of Caspase- and Granzyme B-cleavage sites in protein sequences

    PubMed Central

    Backes, Christina; Kuentzer, Jan; Lenhof, Hans-Peter; Comtesse, Nicole; Meese, Eckart

    2005-01-01

    Caspases and granzyme B are proteases that share the primary specificity to cleave at the carboxyl terminal of aspartate residues in their substrates. Both, caspases and granzyme B are enzymes that are involved in fundamental cellular processes and play a central role in apoptotic cell death. Although various targets are described, many substrates still await identification and many cleavage sites of known substrates are not identified or experimentally verified. A more comprehensive knowledge of caspase and granzyme B substrates is essential to understand the biological roles of these enzymes in more detail. The relatively high variability in cleavage site recognition sequence often complicates the identification of cleavage sites. As of yet there is no software available that allows identification of caspase and/or granzyme with cleavage sites differing from the consensus sequence. Here, we present a bioinformatics tool ‘GraBCas’ that provides score-based prediction of potential cleavage sites for the caspases 1–9 and granzyme B including an estimation of the fragment size. We tested GraBCas on already known substrates and showed its usefulness for protein sequence analysis. GraBCas is available at . PMID:15980455

  19. Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

    PubMed Central

    Marissen, Wilfred E.; Lloyd, Richard E.

    1998-01-01

    Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity. PMID:9819442

  20. Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues.

    PubMed

    Droga-Mazovec, Gabriela; Bojic, Lea; Petelin, Ana; Ivanova, Saska; Romih, Rok; Repnik, Urska; Salvesen, Guy S; Stoka, Veronika; Turk, Vito; Turk, Boris

    2008-07-04

    As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.

  1. The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycin induce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage

    PubMed Central

    Cappellini, Alessandra; Chiarini, Francesca; Ognibene, Andrea; McCubrey, James A.; Martelli, Alberto M.

    2009-01-01

    Resistance to multiple chemotherapeutic agents is a common clinical problem which can arise during cancer treatment. Drug resistance often involves overexpression of the multidrug resistance MDR1 gene, encoding P-glycoprotein (P-gp), a 170-kDa glycoprotein belonging to the ATP-binding cassette superfamily of membrane transporters. We have recently demonstrated apoptosis-induced, caspase-3-dependent P-gp cleavage in human T-lymphoblastoid CEM-R VBL100 cells. However, P-gp contain many aspartate residues which could be targeted by caspases other than caspase-3. To test whether other caspases could cleave P-gp in vivo, we investigated the fate of P-gp during roscovitine- and sangivamycin- induced apoptosis in MCF7 human breast cancer cells, as they lack functional caspase-3. MCF7 cells were stably transfected with human cDNA encoding P-gp. P-gp was cleaved in vitro by purified recombinant caspase-3, -6 and -7. However, P-gp cleavage was not detected in vivo in MCF7 cells induced to undergoing apoptosis by either roscovitine or sangivamycin, despite activation of both caspase-6 and -7. Interestingly, P-gp overexpressing MCF7 cells were more sensitive to either roscovitine or sangivamycin than wild-type cells, suggesting a novel potential therapeutic strategy against P-gp overexpressing cells. Taken together, our results support the concept that caspase-3 is the only caspase responsible for in vivo cleavage of P-gp and also highlight small molecules which could be effective in treating P-gp overexpressing cancers. PMID:19342873

  2. Caspase-3/7-mediated Cleavage of β2-spectrin is Required for Acetaminophen-induced Liver Damage.

    PubMed

    Baek, Hye Jung; Lee, Yong Min; Kim, Tae Hyun; Kim, Joo-Young; Park, Eun Jung; Iwabuchi, Kuniyoshi; Mishra, Lopa; Kim, Sang Soo

    2016-01-01

    The ubiquitously expressed β2-spectrin (β2SP, SPTBN1) is the most common non-erythrocytic member of the β-spectrin gene family. Loss of β2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of β2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that β2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of β2-spectrin cleavage robustly attenuated β2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of β2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of β2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of β2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration. β2-Spectrin, a TGF-β mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of β2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-β in liver damage.

  3. Caspase-3/7-mediated Cleavage of β2-spectrin is Required for Acetaminophen-induced Liver Damage

    PubMed Central

    Baek, Hye Jung; Lee, Yong Min; Kim, Tae Hyun; Kim, Joo-Young; Park, Eun Jung; Iwabuchi, Kuniyoshi; Mishra, Lopa; Kim, Sang Soo

    2016-01-01

    The ubiquitously expressed β2-spectrin (β2SP, SPTBN1) is the most common non-erythrocytic member of the β-spectrin gene family. Loss of β2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of β2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that β2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of β2-spectrin cleavage robustly attenuated β2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of β2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of β2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of β2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration. Conclusions: β2-Spectrin, a TGF-β mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of β2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-β in liver damage. PMID:26884715

  4. Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

    PubMed

    Breckenridge, David G; Stojanovic, Marina; Marcellus, Richard C; Shore, Gordon C

    2003-03-31

    Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

  5. Cleavage of the Bloom’s syndrome gene product during apoptosis by caspase-3 results in an impaired interaction with topoisomerase IIIα

    PubMed Central

    Freire, Raimundo; d’Adda di Fagagna, Fabrizio; Wu, Leonard; Pedrazzi, Graziella; Stagljar, Igor; Hickson, Ian D.; Jackson, Stephen P.

    2001-01-01

    In higher eukaryotes, the integration of signals triggered in response to certain types of stress can result in programmed cell death. Central to these events is the sequential activation of a cascade of proteinases known as caspases. The final activated effector caspases of this cascade digest a number of cellular proteins, in some cases increasing their enzymatic activity, in others destroying their function. Of the proteins shown to be targets for caspase-mediated proteolysis, a surprisingly large proportion are proteins involved in the signalling or repair of DNA damage. Here we investigate whether BLM, the product of the gene mutated in Bloom’s syndrome, a human autosomal disease characterised by cancer predisposition and sunlight sensitivity, is cleaved during apoptosis. BLM interacts with topoisomerase IIIα and has been proposed to play an important role in maintaining genomic integrity through its roles in DNA repair and replication. We show that BLM is cleaved during apoptosis by caspase-3 and reveal that the main cleavage site is located at the junction between the N-terminal and central helicase domains of BLM. Proteolytic cleavage by caspase-3 produces a 120 kDa fragment, which contains the intact helicase domain and three smaller fragments, the relative amounts of which depend on time of incubation with caspase-3. The 120 kDa fragment retains the helicase activity of the intact BLM protein. However, its interaction with topoisomerase IIIα is severely impaired. Since the BLM–topoisomerase interaction is believed to be necessary for many of the replication and recombination functions of BLM, we suggest that caspase-3 cleavage of BLM could alter the localisation and/or function of BLM and that these changes may be important in the process of apoptosis. PMID:11470874

  6. Delphinidin sensitizes prostate cancer cells to TRAIL-induced apoptosis, by inducing DR5 and causing caspase-mediated HDAC3 cleavage

    PubMed Central

    Jeon, Hyelin; Sung, Gi-Jun; So, Youngsin; Kim, InKi; Son, JaeKyoung; Lee, Sang-wook; Yoon, Ho-Geun; Choi, Kyung-Chul

    2015-01-01

    TRAIL can induce apoptosis in some cancer cells and is an immune effector in the surveillance and elimination of developing tumors. Yes, some cancers are resistant to TRAIL. Delphinidin, a polyphenolic compound contained in brightly colored fruits and vegetables, has anti-inflammatory, anti-oxidant, and anti-tumorigenic activities. Here we showed that delphinidin sensitized TRAIL-resistant human prostate cancer cells to undergo apoptosis. Cells treated with delphinidin and TRAIL activated the extrinsic and intrinsic pathways of caspase activation. TRAIL-induced apoptosis in prostate cancer cells pretreated with delphinidin was dependent on death receptor 5 (DR5) and downstream cleavage of histone deacetylase 3 (HDAC3). In conclusion, delphinidin sensitizes prostate cancer cells to TRAIL-induced apoptosis by inducing DR5, thus causing caspase-mediated HDAC3 cleavage. Our data reveal a potential way of chemoprevention of prostate cancer by enabling TRAIL-mediated apoptosis. PMID:25991668

  7. Homoharringtonine combined with aclarubicin and cytarabine synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein.

    PubMed

    Cao, Jiang; Feng, Hao; Ding, Ning-Ning; Wu, Qing-Yun; Chen, Chong; Niu, Ming-Shan; Chen, Wei; Qiu, Ting-Ting; Zhu, Hong-Hu; Xu, Kai-Lin

    2016-11-01

    Homoharringtonine combined with aclarubicin and cytarabine (HAA) is a highly effective treatment for acute myeloid leukemia (AML), especially for t(8;21) AML. However, the underlying mechanisms by which HAA kills t(8;21) AML cells remain unclear. In this study, SKNO-1 and Kasumi-1 cells with t(8;21) were used. Compared with individual or pairwise administration of homoharringtonine, aclarubicin, or cytarabine, HAA showed the strongest inhibition of growth and induction of apoptosis in SKNO-1 and Kasumi-1 cells. HAA caused cleavage of the AML1-ETO (AE) oncoprotein to form truncated AE (ΔAE). Pretreatment with the caspase-3 inhibitor caspase-3 inhibitor Q-DEVD-OPh (QDO) not only suppressed HAA-induced apoptosis but also abrogated the cleavage of AE and generation of ΔAE. These results suggest that HAA synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein, thus providing direct evidence for the strong activity of HAA toward t(8;21) AML.

  8. BAP31 and its caspase cleavage product regulate cell surface expression of tetraspanins and integrin-mediated cell survival.

    PubMed

    Stojanovic, Marina; Germain, Marc; Nguyen, Mai; Shore, Gordon C

    2005-08-26

    BAP31, a resident integral protein of the endoplasmic reticulum membrane, regulates the export of other integral membrane proteins to the downstream secretory pathway. Here we show that cell surface expression of the tetraspanins CD9 and CD81 is compromised in mouse cells from which the Bap31 gene has been deleted. CD9 and CD81 facilitate the function of multiprotein complexes at the plasma membrane, including integrins. Of note, BAP31 does not appear to influence the egress of alpha5beta1 or alpha(v)beta3 integrins to the cell surface, but in Bap31-null mouse cells, these integrins are not able to maintain cellular adhesion to the extracellular matrix in the presence of reduced serum. Consequently, Bap31-null cells are sensitive to serum starvation-induced apoptosis. Reconstitution of wild-type BAP31 into these Bap31-null cells restores integrin-mediated cell attachment and cell survival after serum stress, whereas interference with the functions of CD9, alpha5beta1, or alpha(v)beta3 by antagonizing antibodies makes BAP31 cells act similar to Bap31-null cells in these respects. Finally, in human KB epithelial cells protected from apoptosis by BCL-2, the caspase-8 cleavage product, p20 BAP31, inhibits egress of tetraspanin and integrin-mediated cell attachment. Thus, p20 BAP31 can operate upstream of BCL-2 in living cells to influence cell surface properties due to its effects on protein egress from the endoplasmic reticulum.

  9. Dieldrin induces apoptosis by promoting caspase-3-dependent proteolytic cleavage of protein kinase Cdelta in dopaminergic cells: relevance to oxidative stress and dopaminergic degeneration.

    PubMed

    Kitazawa, M; Anantharam, V; Kanthasamy, A G

    2003-01-01

    We previously reported that dieldrin, one of the potential environmental risk factors for development of Parkinson's disease, induces apoptosis in dopaminergic cells by generating oxidative stress. Here, we demonstrate that the caspase-3-dependent proteolytic activation of protein kinase Cdelta (PKCdelta) mediates as well as regulates the dieldrin-induced apoptotic cascade in dopaminergic cells. Exposure of PC12 cells to dieldrin (100-300 microM) results in the rapid release of cytochrome C, followed by the activation of caspase-9 and caspase-3 in a time- and dose-dependent manner. The superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride significantly attenuates dieldrin-induced cytochrome C release, indicating that reactive oxygen species may contribute to the activation of pro-apoptotic factors. Interestingly, dieldrin proteolytically cleaves native PKCdelta into a 41 kDa catalytic subunit and a 38 kDa regulatory subunit to activate the kinase. The dieldrin-induced proteolytic cleavage of PKCdelta and induction of kinase activity are completely inhibited by pretreatment with 50-100 microM concentrations of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), indicating that the proteolytic activation of PKCdelta is caspase-3-dependent. Additionally, Z-VAD-FMK, Z-DEVD-FMK or the PKCdelta specific inhibitor rottlerin almost completely block dieldrin-induced DNA fragmentation. Because dieldrin dramatically increases (40-80-fold) caspase-3 activity, we examined whether proteolytically activated PKCdelta amplifies caspase-3 via positive feedback activation. The PKCdelta inhibitor rottlerin (3-20 microM) dose-dependently attenuates dieldrin-induced caspase-3 activity, suggesting positive feedback activation of caspase-3 by PKCdelta. Indeed, delivery of catalytically active recombinant PKCdelta via a protein delivery system significantly

  10. Caspase-mediated cleavage of Beclin1 inhibits autophagy and promotes apoptosis induced by S1 in human ovarian cancer SKOV3 cells.

    PubMed

    Li, Xiaoning; Su, Jing; Xia, Meihui; Li, Hongyan; Xu, Ye; Ma, Chunhui; Ma, Liwei; Kang, Jingsong; Yu, Huimei; Zhang, Zhichao; Sun, Liankun

    2016-02-01

    S1, a novel BH3 mimetic, can induce apoptosis dependent on Bax/Bak through inhibition of Bcl-2 in various tumors. S1 also induces autophagy through interrupting the interaction of Bcl-2 and Beclin1. Our results showed that S1 induces apoptosis in human ovarian cancer SKOV3 cells in a time- and dose-dependent manner. Autophagy precedes apoptosis, in SKOV3 cells treated with S1 (6 μmol/L), autophagy reached the maximum peak at 12 h after treatment and decreased to 24 h. In SKOV3 cells treated with different concentrations of S1 for 24 h, the highest level of autophagy was observed with 5 μmol/L and decreased to 10 μmol/L. Autophagy inhibitors 3-MA and CQ enhanced apoptosis induced by S1 in SKOV3 cells. However, overactivation of caspases in apoptosis induced by S1 may inhibit the autophagy-inducing function of Beclin1. Because the pan-caspase inhibitor Z-VAD recovered the autophagy-inducing function of Beclin1 through reduction of activated caspase-mediated cleavage of Beclin1. Furthermore, the Beclin1 cleavage products could further increase apoptosis induced by S1 in SKOV3 cells. This indicates that apoptosis induced by high doses and long exposure of S1 causes the overactivation of caspases and subsequent cleavage of Beclin1, and inhibits the protection of autophagy. Moreover, the cleaved product of Beclin1 further promotes apoptosis induced by S1 in SKOV3 cells. Our results suggest this may be a molecular mechanism for enhancing the sensitivity of cancer cells to apoptosis induced by small molecular compound targeting Bcl-2.

  11. Structural analysis of disease-related TDP-43 D169G mutation: linking enhanced stability and caspase cleavage efficiency to protein accumulation

    PubMed Central

    Chiang, Chien-Hao; Grauffel, Cédric; Wu, Lien-Szu; Kuo, Pan-Hsien; Doudeva, Lyudmila G.; Lim, Carmay; Shen, Che-Kun James; Yuan, Hanna S.

    2016-01-01

    The RNA-binding protein TDP-43 forms intracellular inclusions in amyotrophic lateral sclerosis (ALS). While TDP-43 mutations have been identified in ALS patients, how these mutations are linked to ALS remains unclear. Here we examined the biophysical properties of six ALS-linked TDP-43 mutants and found that one of the mutants, D169G, had higher thermal stability than wild-type TDP-43 and that it was cleaved by caspase 3 more efficiently, producing increased levels of the C-terminal 35 kD fragments (TDP-35) in vitro and in neuroblastoma cells. The crystal structure of the TDP-43 RRM1 domain containing the D169G mutation in complex with DNA along with molecular dynamics simulations reveal that the D169G mutation induces a local conformational change in a β turn and increases the hydrophobic interactions in the RRM1 core, thus enhancing the thermal stability of the RRM1 domain. Our results provide the first crystal structure of TDP-43 containing a disease-linked D169G mutation and a disease-related mechanism showing that D169G mutant is more susceptible to proteolytic cleavage by caspase 3 into the pathogenic C-terminal 35-kD fragments due to its increased stability in the RRM1 domain. Modulation of TDP-43 stability and caspase cleavage efficiency could present an avenue for prevention and treatment of TDP-43-linked neurodegeneration. PMID:26883171

  12. Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: IMPLICATIONS FOR ALLOGRAFT REJECTION.

    PubMed

    Burzynski, Laura C; Humphry, Melanie; Bennett, Martin R; Clarke, Murray C H

    2015-10-09

    Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death

    PubMed Central

    Rogers, Corey; Fernandes-Alnemri, Teresa; Mayes, Lindsey; Alnemri, Diana; Cingolani, Gino; Alnemri, Emad S.

    2017-01-01

    Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and progression to secondary necrosis, and provide a molecular mechanism for secondary necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are dependent on caspase activation, we propose that they are forms of programmed necrosis. PMID:28045099

  14. Bioluminescent imaging of TRAIL-induced apoptosis through detection of caspase activation following cleavage of DEVD-aminoluciferin.

    PubMed

    Liu, Jue Judy; Wang, Wenge; Dicker, David T; El-Deiry, Wafik S

    2005-08-01

    Apoptosis, the most common and well-defined form of programmed cell death (PCD), is often impaired in cancer and neurodegenerative diseases and can limit conventional therapy. Bioluminescent molecular imaging was employed to study apoptosis in human colon cancer cells that have been treated with various doses of the therapeutic agent TRAIL (tumor necrosis factor-related apoptosis inducing ligand). While monitoring therapeutic response through a proluminescent, caspase-activated DEVD-aminoluciferin reagent (Caspase-Glo 3/7) which produced strong, stable signals, alternate preparations of the reagent were explored for non-invasive imaging methods. Dissolving the lyophilized DEVD-aminoluciferin compound in Dulbecco's PBS instead of lysis buffer (along with heat inactivation of an accompanying exogenous luciferase protein by heating at 85 degrees C for 20 minutes) yielded a minimally invasive apoptosis detector, with maximum luminescence intensities 2.5-fold stronger than those produced by D-luciferin at a final concentration of 100 microg/mL. Bioluminescent imaging of cancer therapeutic response through minimally invasive detection of caspase activation may serve as an important tool in monitoring apoptosis in vivo and in vitro.

  15. Grb7 and Hax1 may colocalize partially to mitochondria in EGF-treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1.

    PubMed

    Qian, Lei; Bradford, Andrew M; Cooke, Peter H; Lyons, Barbara A

    2016-07-01

    Growth factor receptor bound protein 7 (Grb7) is a signal-transducing adaptor protein that mediates specific protein-protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein-binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras-GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti-apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor-treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time-dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  16. Differential proteomic analysis of HL60 cells treated with secalonic acid F reveals caspase 3-induced cleavage of Rho GDP dissociation inhibitor 2.

    PubMed

    Li, Ning; Yi, Zhiwei; Wang, Yuqiao; Zhang, Qin; Zhong, Tianhua; Qiu, Yinkun; Wu, Zhen; Tang, Xixiang

    2012-12-01

    Secalonic acid F (SAF) has been previously identified, however, little is known about its cytotoxic activity and related cytotoxic mechanism. The aim of this study was to evaluate the cytotoxic activity of SAF isolated from a deep sea originated fungus Penicillium sp. F11 in HL60 cells and to analyze the differences in protein expression of HL60 cells treated with SAF. The CCK-8 assay and Annexin V-FLUOS/PI assay indicated that SAF displayed dose- and time-dependent inhibition of HL60 cell proliferation and induced apoptosis. Two-dimensional gel electrophoresis (2-DE) analysis of HL60 cells treated with SAF (4 µg/ml) revealed 10 differentially expressed protein spots (P<0.05), 5 upregulated and 5 downregulated. Three spots (1 downregulated and 2 upregulated) were identified as Rho GDP dissociation inhibitor 2 (RhoGDI 2) proteins by MALDI-TOF MS. Western blotting further demonstrated the decreased abundance of full-length RhoGDI 2 together with the increased abundance of caspase 3-cleaved product of RhoGDI 2. The caspase 3 inhibitor Ac-DEVD-CHO could suppress the cytotoxic effect of SAF and significantly block the cleavage of RhoGDI 2. RhoGDI 2 is a cytosolic regulator of Rho GTPase and the caspase 3-cleaved product of RhoGDI 2 can advance progression of the apoptotic process. Our data showed that SAF may modulate RhoGDI 2 levels in HL60 cells, thereby potentially disrupting cell signaling pathways important for HL60 cell function.

  17. Identification of a post-translationally myristoylated autophagy-inducing domain released by caspase cleavage of Huntingtin

    PubMed Central

    Martin, Dale D.O.; Heit, Ryan J.; Yap, Megan C.; Davidson, Michael W.; Hayden, Michael R.; Berthiaume, Luc G.

    2014-01-01

    Huntington disease (HD) is a debilitating neurodegenerative disease characterized by the loss of motor control and cognitive ability that ultimately leads to death. It is caused by the expansion of a polyglutamine tract in the huntingtin (HTT) protein, which leads to aggregation of the protein and eventually cellular death. Both the wild-type and mutant form of the protein are highly regulated by post-translational modifications including proteolysis, palmitoylation and phosphorylation. We now demonstrate the existence of a new post-translational modification of HTT: the addition of the 14 carbon fatty acid myristate to a glycine residue exposed on a caspase-3-cleaved fragment (post-translational myristoylation) and that myristoylation of this fragment is altered in a physiologically relevant model of mutant HTT. Myristoylated HTT553–585–EGFP, but not its non-myristoylated variant, initially localized to the ER, induced the formation of autophagosomes and accumulated in abnormally large autophagolysosomal/lysosomal structures in a variety of cell types, including neuronal cell lines under nutrient-rich conditions. Our results suggest that accumulation of myristoylated HTT553–586 in cells may alter the rate of production of autophagosomes and/or their clearance through the heterotypic autophagosomal/lysosomal fusion process. Overall, our novel observations establish a role for the post-translational myristoylation of a caspase-3-cleaved fragment of HTT, highly similar to the Barkor/ATG14L autophagosome-targeting sequence domain thought to sense, maintain and/or promote membrane curvature in the regulation of autophagy. Abnormal processing or production of this myristoylated HTT fragment might be involved in the pathophysiology of HD. PMID:24459296

  18. Caspase 3 activation and PARP cleavage in lymphocytes from newborn babies of diabetic mothers with unbalanced glycaemic control.

    PubMed

    Tarquini, F; Tiribuzi, R; Crispoltoni, L; Porcellati, S; Del Pino, A M; Orlacchio, A; Coata, G; Arnone, S; Torlone, E; Cappuccini, B; Di Renzo, G C; Orlacchio, A

    2014-01-01

    Several epidemiological studies showed that gestational diabetes mellitus is the most frequent metabolic disorder of pregnancy, the pathogenesis of which has yet to be completely clarified. The aim of this study was to investigate the presence and processing of caspase 3 (Casp3) and poly(ADP-ribose) polymerase 1 (PARP1) in cord blood lymphocytes as markers of apoptosis in relation to glycaemic control during intrauterine life. Our results showed a specific positive correlation between the levels of active Casp3 (17-19 kDa) and the inactive form of PARP1 (89 kDa) in lymphocytes isolated from newborn babies of diabetic women with unbalanced glycaemic control, with a direct correlation between the activation of casp3 and the inactivation of PARP1, that makes lymphocytes unresponsive towards lipopolysaccharide stimulation, highlighting an altered functional response. Besides more studies are required to fully correlate the activation of the apoptotic process during the intrauterine life with the foetal health later in life, our study indicates that a cord blood lymphocyte, an easily accessible source, is informative about the activation of apoptotic stimuli in circulating cells of newborn babies in relation to the glycaemic control reached by the mother during pregnancy.

  19. M10, a Caspase Cleavage Product of the Hepatocyte Growth Factor Receptor, Interacts with Smad2 and Demonstrates Anti-Fibrotic Properties in Vitro and in Vivo

    PubMed Central

    Atanelishvili, Ilia; Shirai, Yuichiro; Akter, Tanjina; Buckner, Taylor; Noguchi, Atsushi; Silver, Richard M.; Bogatkevich, Galina S.

    2016-01-01

    Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif “DEVD-T” that upon cleavage by caspase-3 generates a 10 amino acid peptide, TRPASFWETS, designated as “M10”. M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFβ-stimulated normal lung and skin fibroblasts. M10 interacts with the MH2 domain of Smad2 and inhibits TGFβ-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases. PMID:26772959

  20. M10, a caspase cleavage product of the hepatocyte growth factor receptor, interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo.

    PubMed

    Atanelishvili, Ilia; Shirai, Yuichiro; Akter, Tanjina; Buckner, Taylor; Noguchi, Atsushi; Silver, Richard M; Bogatkevich, Galina S

    2016-04-01

    Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif "DEVD-T" that on cleavage by caspase-3 generates a 10-amino acid peptide, TRPASFWETS, designated as "M10". M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFβ-stimulated normal lung and skin fibroblasts. M10 interacts with the Mad Homology 2 domain of Smad2 and inhibits TGFβ-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases.

  1. The novel, orally bioavailable HSP90 inhibitor NVP-HSP990 induces cell cycle arrest and apoptosis in multiple myeloma cells and acts synergistically with melphalan by increased cleavage of caspases.

    PubMed

    Lamottke, Britta; Kaiser, Martin; Mieth, Maren; Heider, Ulrike; Gao, Zhenhai; Nikolova, Zariana; Jensen, Michael R; Sterz, Jan; von Metzler, Ivana; Sezer, Orhan

    2012-05-01

    Heat shock protein 90 (HSP90) binds and stabilizes numerous proteins and kinases essential for myeloma cell survival and proliferation. We and others have recently demonstrated that inhibition of HSP90 by small molecular mass inhibitors induces cell death in multiple myeloma (MM). However, some of the HSP90 inhibitors involved in early clinical trials have shown limited antitumor activity and unfavorable toxicity profiles. Here, we analyzed the effects of the novel, orally bioavailable HSP90 inhibitor NVP-HSP990 on MM cell proliferation and survival. The inhibitor led to a significant reduction in myeloma cell viability and induced G2 cell cycle arrest, degradation of caspase-8 and caspase-3, and induction of apoptosis. Inhibition of the HSP90 ATPase activity was accompanied by the degradation of MM phospho-Akt and phospho-ERK1/2 and upregulation of Hsp70. Exposure of MM cells to a combination of NVP-HSP990 and either melphalan or histone deacetylase (HDAC) inhibitors caused synergistic inhibition of viability, increased induction of apoptosis, and was able to overcome the primary resistance of the cell line RPMI-8226 to HSP90 inhibition. Combined incubation with melphalan and NVP-HSP990 led to synergistically increased cleavage of caspase-2, caspase-9, and caspase-3. These data demonstrate promising activity for NVP-HSP990 as single agent or combination treatment in MM and provide a rationale for clinical trials.

  2. FoxO3a governs early microglial proliferation and employs mitochondrial depolarization with caspase 3, 8, and 9 cleavage during oxidant induced apoptosis.

    PubMed

    Shang, Yan Chen; Chong, Zhao Zhong; Hou, Jinling; Maiese, Kenneth

    2009-11-01

    Microglia of the central nervous system have a dual role in the ability to influence the survival of neighboring cells. During inflammatory cell activation, microglia can lead to the disposal of toxic cellular products and permit tissue regeneration, but microglia also may lead to cellular destruction with phagocytic removal. For these reasons, it is essential to elucidate not only the underlying pathways that control microglial activation and proliferation, but also the factors that determine microglial survival. In this regard, we investigated in the EOC 2 microglial cell line with an oxygen-glucose deprivation (OGD) injury model of oxidative stress the role of the "O" class forkhead transcription factor FoxO3a that in some scenarios is closely linked to immune system function. We demonstrate that FoxO3a is a necessary element in the control of early and late apoptotic injury programs that involve membrane phosphatidylserine externalization and nuclear DNA degradation, since transient knockdown of FoxO3a in microglia preserves cellular survival 24 hours following OGD exposure. However, prior to the onset of apoptotic injury, FoxO3a facilitates the activation and proliferation of microglia as early as 3 hours following OGD exposure that occurs in conjunction with the trafficking of the unphosphorylated and active post-translational form of FoxO3a from the cytoplasm to the cell nucleus. FoxO3a also can modulate apoptotic mitochondrial signal transduction pathways in microglia, since transient knockdown of FoxO3a prevents mitochondrial membrane depolarization as well as the release of cytochrome c during OGD. Control of this apoptotic cascade also extends to progressive caspase activation as early as 1 hour following OGD exposure. The presence of FoxO3a is necessary for the expression of cleaved (active) caspase 3, 8, and 9, since loss of FoxO3a abrogates the induction of caspase activity. Interestingly, elimination of FoxO3a reduced caspase 9 activity to a lesser

  3. Increased anticancer activity of the thymidylate synthase inhibitor BGC9331 combined with the topoisomerase I inhibitor SN-38 in human colorectal and breast cancer cells: induction of apoptosis and ROCK cleavage through caspase-3-dependent and -independent mechanisms.

    PubMed

    Coudray, Anne-Marie; Louvet, Christophe; Kornprobst, Michel; Raymond, Eric; André, Thierry; Tournigand, Christophe; Faivre, Sandrine; De Gramont, Aimery; Larsen, Annette K; Gespach, Christian

    2005-08-01

    The folate analogue BGC9331 is a new thymidylate synthase (TS) inhibitor showing a broad spectrum of cyto-toxic activity against several human solid tumors, including colorectal cancer. In this study, we investigated the anticancer activity of BGC9331 either alone or combined with 5-fluorouracil (5-FU), MTA (multi-target antifolate), oxali-platin and SN-38, the active metabolite of the topoisomerase I inhibitor CPT-11. The antiproliferative activity of each drug and BGC9331-based combinations was investigated in the HT-29 human colorectal cancer cell line and its HT-29/5-FU counterparts selected for resistance to 5-FU. BGC9331 combined with MTA or SN-38 induced synergistic responses in HT-29 cells. Treatment of HT-29 cells with either BGC9331 or SN-38 increased caspase-3 activity and the percentage of apoptotic cells from 3 to 13%. Both drugs also augmented the proteolytic cleavage of the Rho-kinase ROCK-1 that was attenuated by the caspase-3 pathway inhibitor z-DEVD-fmk. BGC9331 combined with SN-38 further increased the percentage of apoptotic cells to 25%, and inhibited cell cycle progression and cell proliferation by 65%. This was accompanied by proteolytic activation of ROCK-1, through both caspase-3-dependent and -independent mechanisms, as shown in caspase-3-deficient MCF-7 breast cancer cells. These encouraging results warrant further preclinical investigations and clinical trials on the use of BGC9331 combined with SN-38/CPT-11 in treatment of patients with advanced colorectal or gastric cancers.

  4. In vivo proof-of-concept of removal of the huntingtin caspase cleavage motif-encoding exon 12 approach in the YAC128 mouse model of Huntington's disease.

    PubMed

    Casaca-Carreira, João; Toonen, Lodewijk J A; Evers, Melvin M; Jahanshahi, Ali; van-Roon-Mom, Willeke M C; Temel, Yasin

    2016-12-01

    Huntington's disease (HD) is a progressive autosomal dominant disease, caused by a CAG repeat expansion in the HTT gene, resulting in an expanded polyglutamine stretch at the N-terminal of the huntingtin protein. An important event in HD pathogenesis appears to be the proteolysis of the mutant protein, which forms N-terminal huntingtin fragments. These fragments form insoluble aggregates and are found in nuclei and cytoplasm of affected neurons where they interfere with normal cell functioning. Important cleavage sites are encoded by exon 12 of HTT. A novel approach is Htt protein modification through exon skipping, which has recently been proven effective both in vitro and in vivo. Here we report proof-of-concept of AON 12.1 in vivo using the YAC128 mouse model of HD. Our results support and encourage future longitudinal studies exploring the therapeutic effects of sustained infusions in the YAC128 mouse model.

  5. L-BMAA induced ER stress and enhanced caspase 12 cleavage in human neuroblastoma SH-SY5Y cells at low nonexcitotoxic concentrations.

    PubMed

    Okle, Oliver; Stemmer, Kerstin; Deschl, Ulrich; Dietrich, Daniel R

    2013-01-01

    The cyanobacterial β-N-methylamino-L-alanine (L-BMAA) is described as a low-potency excitotoxin, possibly a factor in the increased incidence of amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia complex (PDC) in Guam. The latter association is intensively disputed, as L-BMAA concentrations required for toxic effects exceed those assumed to occur via food. The question thus was raised whether L-BMAA leads to neurodegeneration at nonexcitotoxic conditions. Using human SH-SY5Y neuroblastoma cells, L-BMAA-transport, incorporation into proteins, and subsequent impairment of cellular protein homeostasis were investigated. Binding of L-BMAA to intracellular proteins, but no clear protein incorporation was detected in response to (14)C-L-BMAA exposures. Nevertheless, low L-BMAA concentrations (≥ 0.1mM, 48 h) increased protein ubiquitination, 20S proteasomal and caspase 12 activity, expression of the endoplasmic reticulum (ER) stress marker CHOP, and enhanced phosphorylation of elf2α in SH-SY5Y cells. In contrast, high L-BMAA concentrations (≥ 1mM, 48 h) increased reactive oxygen species and protein oxidization, which were partially ameliorated by coincubation with vitamin E. L-BMAA-mediated cytotoxicity was observable 48 h following ≥ 2mM L-BMAA treatment. Consequently, the data presented here suggest that low L-BMAA concentrations result in a dysregulation of the cellular protein homeostasis with ensuing ER stress that is independent from high-concentration effects such as excitotoxicity and oxidative stress. Thus, the latter could be a contributing factor in the onset and slow progression of ALS/PDC in Guam.

  6. A comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways.

    PubMed

    Shekhawat, Sujan S; Campbell, Sean T; Ghosh, Indraneel

    2011-10-17

    Caspases play a central role in apoptosis, differentiation, and proliferation, and represent important therapeutic targets for treating cancer and inflammatory disorders. Toward the goal of developing new tools to probe caspase substrate cleavage specificity as well as to systematically interrogate caspase activation pathways, we have constructed and investigated a comprehensive panel of caspase biosensors with a split-luciferase enabled bioluminescent read out. We first interrogated the panel of caspase biosensors for substrate cleavage specificity of caspase 1-10 in widely utilized in vitro translation systems, namely, rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE). Commercial RRL was found to be unsuitable for investigating caspase specificity, owing to surprising levels of endogenous caspase activity, while specificity profiles of the caspase sensors in WGE agree very well with traditional peptide probes. The full panel of biosensors was utilized for studying caspase activation and inhibition in several mammalian cytosolic extracts, clearly demonstrating that they can be utilized to directly monitor activation or inhibition of procaspase 3/7. Furthermore, the complete panel of caspase biosensors also provided new insights into caspase activation pathways wherein we surprisingly discovered the activation of procaspase 3/7 by caspase 4/5.

  7. Typhonium flagelliforme induces apoptosis in CEMss cells via activation of caspase-9, PARP cleavage and cytochrome c release: its activation coupled with G0/G1 phase cell cycle arrest.

    PubMed

    Mohan, Syam; Abdul, Ahmad Bustamam; Abdelwahab, Siddig Ibrahim; Al-Zubairi, Adel S; Sukari, Mohamed Aspollah; Abdullah, Rasedee; Elhassan Taha, Manal Mohamed; Ibrahim, Mohamed Yousif; Syam, Suvitha

    2010-10-05

    The plant Typhonium flagelliforme (TF), commonly known as 'rodent tuber' in Malaysia, is often used as traditional remedy for cancer, including leukemia. We had previously identified morphologically that the linoleic acid rich fraction (DCM/F7) from the tubers of this plant induces selective anti-proliferative effects and apoptosis in CEMss cells. In this present study, we subjected the same DCM/F7 fraction to cell based activity analyses in order to determine the possible mechanism of cell death in leukemic CEMss cells in vitro. Extraction of Typhonium flagelliforme tuber has done and fractionation has been done by vacuum liquid column chromatography. The anti-proliferative activity was assayed using MTT and the apoptosis detection was done by Annexin V and DNA laddering assay. Colorimetric caspase assay and immunoblot analysis were employed to detect the expression of protein associated with cell death. Cell cycle analysis was done using flow cytometry. We found that the cancer inhibitory effect of the DCM/F7 fraction in CEMss cells was 3 ± 0.08 μg/ml (IC(50)). An early apoptotic induction in CEMss cells was observed by Annexin V assay, which showed a clear dose-dependent DNA fragmentation being observed in gel electrophoresis at 10 and 20 μg/ml. The DCM/F7 fraction at 3 μg/ml significantly arrested CEMss cells at G0/G1 phase (p<0.05). A constant but increasing pattern-related Sub-G0/G1 index was observed between 12 and 72 h treatment. In relation to this, we further investigated the biochemical events leading to cell death and found that the DCM/F7 fraction increased the cellular levels of caspase-3 and -9 on treated cells. Our results indicated that cytochrome c from mitochondria into the cytosol increased gradually as the DCM/F7 concentration increases, which later lead to the subsequent cleavage of PARP in to 85kDa fragments. On the contrary, Bcl-2 protein was found to decrease concomitantly during treatment. Collectively, results presented in this study

  8. Distinct MAPK signaling pathways, p21 up-regulation and caspase-mediated p21 cleavage establishes the fate of U937 cells exposed to 3-hydrogenkwadaphnin: Differentiation versus apoptosis

    SciTech Connect

    Moosavi, Mohammad Amin; Yazdanparast, Razieh

    2008-07-01

    Despite the depth of knowledge concerning the pathogenesis of acute myeloblastic leukemia (AML), long-term survival remains unresolved. Therefore, new agents that act more selectively and more potently are required. In that line, we have recently characterized a novel diterpene ester, called 3-hydrogenkwadaphnin (3-HK), with capability to induce both differentiation and apoptosis in various leukemia cell lines. These effects of 3-HK were mediated through inhibition of inosine 5'-monophosphate dehydrogenase, a selective up-regulated enzyme in cancerous cells, especially leukemia. However, it remains elusive to understand how cells display different fates in response to 3-HK. Here, we report the distinct molecular signaling pathways involved in forcing of 3-HK-treated U937 cells to undergo differentiation and apoptosis. After 3-HK (15 nM) treatment, a portion of U937 cells adhered to the culture plates and showed macrophage criteria while others remained in suspension and underwent apoptosis. The differentiated cells arrested in G{sub 0}/G{sub 1} phase of cell cycle and showed early activation of ERK1/2 pathway (3 h) along with ERK-dependent p21{sup Cip/WAF1} (p21) up-regulation and expression of p27{sup Kip1} and Bcl-2. In contrast, the suspension cells underwent apoptosis through Fas/FasL and mitochondrial pathways. The occurrence of apoptosis in these cells were accompanied with caspase-8-mediated p21 cleavage and delayed activation (24 h) of JNK1/2 and p38 MAPK. Taken together, these results suggest that distinct signaling pathways play a pivotal role in fates of drug-treated leukemia cells, thus this may pave some novel therapeutical utilities.

  9. Inclusion Complex of Zerumbone with Hydroxypropyl-β-Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio

    PubMed Central

    Abdul, Ahmad Bustamam; Sukari, Mohd Aspollah; Abdelwahab, Siddig Ibrahim; Eid, Eltayeb E. M.; Kamalidehghan, Behnam; Anasamy, Theebaa; Ng, Kuan Beng; Syam, Suvitha; Arbab, Ismail Adam; Rahman, Heshu Sulaiman; Ali, Hapipah Mohd

    2013-01-01

    Zerumbone (ZER) isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl-β-cyclodextrin (HPβCD) to enhance ZER's solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HPβCD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HPβCD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HPβCD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans. PMID:23737847

  10. Caspase 12 in calnexin-deficient cells.

    PubMed

    Groenendyk, Jody; Zuppini, Anna; Shore, Gordon; Opas, Michal; Bleackley, R Chris; Michalak, Marek

    2006-11-07

    We investigated a role for calnexin, caspase 12, and Bap31 in endoplasmic reticulum stress-induced apoptosis in calnexin-deficient mouse embryonic fibroblasts and a calnexin-deficient human T cell line (NKR). We showed that calnexin-deficient mouse embryonic fibroblasts are relatively resistant to endoplasmic reticulum stress-induced apoptosis. Western blot analysis demonstrated that both wild-type and calnexin-deficient cells contained a caspase 12 protein. Caspase 12 expression was slightly inhibited in calnexin-deficient cells, and the protein carried out specific cleavage in the presence of thapsigargin. Immunoprecipitation experiments revealed that in the endoplasmic reticulum, caspase 12 forms complexes with Bap31 and calnexin. Treatment of wild-type cells with thapsigargin induced apoptosis and cleavage of Bap31. However, in the absence of calnexin, there was no significant cleavage of Bap31. There was also a negligible processing of caspase 8 in these cells. This work indicates that calnexin may play a role in modulating the sensitivity of a cell to apoptosis induced by endoplasmic reticulum stress, in conjunction with caspase 12 and Bap31.

  11. Nimesulide, a selective COX-2 inhibitor, acts synergistically with ionizing radiation against A549 human lung cancer cells through the activation of caspase-8 and caspase-3.

    PubMed

    Kim, Byeong Mo; Won, Juyoon; Maeng, Kyung Ah; Han, Young Soo; Yun, Yeon-Sook; Hong, Sung Hee

    2009-05-01

    Several lines of evidence suggest that non-steroidal anti-inflammatory drugs (NSAIDs) have a radiosensitizing effect on cancer cells in vitro and in vivo, but little is known about the underlying cellular mechanism. In this study, we found that the treatment with the NSAID nimesulide significantly increased the sensitivity of A549 human non-small cell lung cancer cells to radiotherapy. The combined nimesulide-radiation treatment increased apoptosis, induced the cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP), activated caspase-8, and induced cleavage of Bid. A pan-caspase inhibitor, z-VAD-fmk, suppressed this increase in apoptosis and also suppressed the cleavage of caspase-8, caspase-3, and PARP, suggesting a caspase-dependent mechanism. In addition, z-IETD-fmk, a selective caspase-8 inhibitor, suppressed the nimesulide- and radiation-induced cleavage activation of caspase-9, caspase-3, caspase-8, and Bid, and suppressed the concomitant apoptosis, indicating that the nimesulide-induced increase in radiosensitivity was initiated by caspase-8. However, the caspase-3 inhibitor z-DEVD-fmk failed to suppress activation of the caspase-8/Bid pathway, indicating that caspase-3 activation occurred downstream of caspase-8 activation in our experiments. Marked antitumor effects, which were evaluated by measuring protracted tumor regression, were observed when nude mice were treated with a combination of nimesulide at a clinically achievable dose (0.5 mg/kg) and radiation therapy. Our results, demonstrating the radiosensitivity-increasing and tumor growth-inhibiting effects of nimesulide, suggest that nimesulide may be suitable as an adjuvant to enhance the efficacy and selectivity of radiotherapy.

  12. Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach.

    PubMed

    Ai, X; Butts, B; Vora, K; Li, W; Tache-Talmadge, C; Fridman, A; Mehmet, H

    2011-09-01

    Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity.

  13. Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach

    PubMed Central

    Ai, X; Butts, B; Vora, K; Li, W; Tache-Talmadge, C; Fridman, A; Mehmet, H

    2011-01-01

    Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity. PMID:21881607

  14. Specific Cleavage of the Nucleoprotein of Fish Rhabdovirus.

    PubMed

    Zhou, G-Z; Yi, Y-J; Chen, Z-Y; Zhang, Q-Y

    2015-11-01

    Siniperca chuatsi rhabdovirus (SCRV) is one of myriad rhabdoviruses recorded in fish. Preliminary data show that inhibition of the SCRV nucleoprotein (N) could significantly reduce the progeny virus titers in infected Epithelioma papulosum cyprinid (EPC) cells. Here, the authors propose that cleavage of the viral 47-kDa N protein is caspase-mediated based on caspase inhibition experiments, transient expression in EPC transfection, and analysis of cleavage sites. Cleavage of the SCRV N protein in culture was prevented by a pan-caspase inhibitor, z-VAD-FMK (z-Val-Ala-DL-Asp-fluoromethyl ketone). Subsequently, N was transiently expressed in EPC cells, the results of which indicated that the specific cleavage of N also occurred in the cells transfected with N-GFP plasmid. Several truncated fragments of the N gene were constructed and transiently transfected into EPC cells. Immunoblotting results indicated that D324 and D374 are the cleavage sites of N by caspases. The authors also found that z-VAD-FMK could inhibit the cytopathic effect in SCRV-infected EPC cells but not affect the production of infectious progeny, suggesting that the caspase-mediated cleavage of N protein is not required for in vitro SCRV replication. To the authors' knowledge, this is the first report on the cleavage of rhabdovirus proteins. © The Author(s) 2015.

  15. Oxidative modification of caspase-9 facilitates its activation via disulfide-mediated interaction with Apaf-1.

    PubMed

    Zuo, Yong; Xiang, Binggang; Yang, Jie; Sun, Xuxu; Wang, Yumei; Cang, Hui; Yi, Jing

    2009-04-01

    Intracellular reactive oxygen species (ROS) are known to regulate apoptosis. Activation of caspase-9, the initial caspase in the mitochondrial apoptotic cascade, is closely associated with ROS, but it is unclear whether ROS regulate caspase-9 via direct oxidative modification. The present study aims to elucidate the molecular mechanisms by which ROS mediate caspase-9 activation. Our results show that the cellular oxidative state facilitates caspase-9 activation. Hydrogen peroxide treatment causes the activation of caspase-9 and apoptosis, and promotes an interaction between caspase-9 and apoptotic protease-activating factor 1 (Apaf-1) via disulfide formation. In addition, in an in vitro mitochondria-free system, the thiol-oxidant diamide promotes auto-cleavage of caspase-9 and the caspase-9/Apaf-1 interaction by facilitating the formation of disulfide-linked complexes. Finally, a point mutation at C403 of caspase-9 impairs both H(2)O(2)-promoted caspase-9 activation and interaction with Apaf-1 through the abolition of disulfide formation. The association between cytochrome c and the C403S mutant is significantly weaker than that between cytochrome c and wild-type caspase-9, indicating that oxidative modification of caspase-9 contributes to apoptosome formation under oxidative stress. Taken together, oxidative modification of caspase-9 by ROS can mediate its interaction with Apaf-1, and can thus promote its auto-cleavage and activation. This mechanism may facilitate apoptosome formation and caspase-9 activation under oxidative stress.

  16. Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism

    PubMed Central

    Dubey, Megha; Nagarkoti, Sheela; Awasthi, Deepika; Singh, Abhishek K; Chandra, Tulika; Kumaravelu, J; Barthwal, Manoj K; Dikshit, Madhu

    2016-01-01

    Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway. PMID:27584786

  17. Caspase Exploitation by Legionella pneumophila

    PubMed Central

    Krause, Kathrin; Amer, Amal O.

    2016-01-01

    Legionella pneumophila remains a major health concern, especially for hospitalized patients. L. pneumophila in the environment can survive extracellular or as protozoan parasite within amoeba. After human infection it efficiently replicates in alveolar macrophages without activating inflammasome assembly and cleavage of caspase-1. In contrast murine macrophages actively recognize intracellular L. pneumophila via inflammasome components which initiate pro-inflammatory cytokine secretion, phagosomal maturation and pyroptotic cell death thereby leading to bacterial restriction. During this process flagellin-dependent and -independent signaling pathways trigger the canonical as well as the non-canonical inflammasome. This review describes the current knowledge about L. pneumophila-induced inflammasome pathways in permissive and restrictive host cells. PMID:27148204

  18. Caspases cleave and inhibit the microRNA processing protein DiGeorge Critical Region 8.

    PubMed

    Gong, Ming; Chen, Yanqiu; Senturia, Rachel; Ulgherait, Matthew; Faller, Michael; Guo, Feng

    2012-06-01

    DGCR8 (DiGeorge Critical Region 8) is an essential microRNA (miRNA) processing protein that recognizes primary transcripts of miRNAs (pri-miRNAs) and triggers their cleavage by the Drosha nuclease. We previously found that Fe(III) heme binds and activates DGCR8. Here we report that in HeLa cells, DGCR8 undergoes two proteolytic events that produce two C-terminal fragments called DGCR8(C1) and DGCR8(C2) , respectively. DGCR8(C2) accumulates during apoptosis and is generated through cleavage by a caspase. The caspase cleavage site is located in the central loop of the heme-binding domain. Cleavage of DGCR8 by caspase-3 in vitro results in loss of the otherwise tightly bound Fe(III) heme cofactor, dissociation of the N- and C-terminal proteolytic fragments, and inhibition of the pri-miRNA processing activity. These results reveal an intrinsic mechanism in the DGCR8 protein that seems to have evolved for regulating miRNA processing via association with Fe(III) heme and proteolytic cleavage by caspases. Decreased expression of miRNAs has been observed in apoptotic cells, and this change was attributed to caspase-mediated cleavage of a down-stream miRNA processing nuclease Dicer. We suggest that both the Drosha and Dicer cleavage steps of the miRNA maturation pathway may be inhibited in apoptosis and other biological processes where caspases are activated.

  19. Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: A role for the IAPs

    SciTech Connect

    Cheung, Herman H.; Lynn Kelly, N.; Liston, Peter; Korneluk, Robert G. . E-mail: bob@mgcheo.med.uottawa.ca

    2006-07-15

    Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to degenerative disorders. An emerging area of study in apoptosis is the critical contribution of the endoplasmic reticulum (ER) in both mitochondrial and ER specific apoptosis pathways. Here we show that brefeldin A and tunicamycin-mediated ER stress lead to caspase-dependent apoptosis involving caspase-2. Confocal microscopy and subcellular fractionation indicate that caspase-2 is localized to the ER, and following ER stress, the processing of caspase-2 and -9 is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). Inhibition and silencing of either caspase-2 or caspase-9 suppress ER stress-induced apoptosis, as demonstrated by annexin V binding. Similarly, transduction with an adenovirus encoding either Inhibitors of Apoptosis (IAP) protein HIAP1/c-IAP2 or HIAP2/c-IAP1 also suppresses ER stress-induced apoptosis. However, among HIAP1, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Our results thus indicate a novel mechanism by which HIAP2 can regulate ER-initiated apoptosis by modulating the activity of caspase-2.

  20. Detection of caspase-activation in intact lymphoid cells using standard caspase substrates and inhibitors.

    PubMed

    Mack, A; Fürmann, C; Häcker, G

    2000-07-31

    Members of the caspase family of proteases are important in the implementation of apoptotic cell death. These caspases are intracellularly activated upon a death stimulus, and exhibit a distinctive proteolytic activity which transmits a death signal and readily detected by measuring the cleavage of synthetic substrates in cell extracts. In this report, we show that apoptosis-associated caspase activation can be recorded not only in cell lysates but also in intact lymphoid cells with commercially available peptides which are either biotinylated or carry an amino-methylcoumarin (AMC) group. Incubation of intact cells induced to undergo apoptosis with Ac-Asp-Glu-Val-Asp-AMC (DEVD-AMC) leads to the release of AMC in amounts very similar to the amounts released when cell extracts are prepared and incubated with DEVD-AMC. This release can be detected by a fluorescence read-out and is blocked by caspase-inhibitors such as Ac-DEVD-cho or Z-VAD-fmk. Similarly, labelling of intact cells with the biotinylated peptides Tyr-Val-Ala-Asp-cmk (YVAD-cmk) or YVAD-faom permits the detection of active caspases by affinity blotting and the detection of apoptotic cells by FACS analysis. These methods enable the investigator to detect at the single-cell level those cells which have activated their caspases and to evaluate such activation without the need for lysis of the cells.

  1. Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

    SciTech Connect

    S Vaidya; E Velazquez-Delgado; G Abbruzzese; J Hardy

    2011-12-31

    Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergo a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.

  2. Implication of caspases and subcellular compartments in tert-butylhydroperoxide induced apoptosis.

    PubMed

    Haidara, Khadidja; Marion, Michel; Gascon-Barré, Marielle; Denizeau, Francine; Averill-Bates, Diana A

    2008-05-15

    Oxidative stress has been implicated in many physiopathologies including neurodegenerative diseases, cancer, cardiovascular and respiratory diseases, and in mechanisms of action of environmental toxicants. tert-butylhydroperoxide (t-BHP) is an organic lipid hydroperoxide analogue, which is commonly used as a pro-oxidant for evaluating mechanisms involving oxidative stress in cells and tissues. This study investigates mechanisms of apoptosis induced by oxidative stress in hepatocytes, in particular, the involvement of caspases and subcellular compartments. Freshly isolated hepatocytes were exposed to 0.4 mM t-BHP during 1 h. A general caspase inhibitor, Boc-D-FMK, reduced t-BHP-induced apoptosis (chromatin condensation), confirming the involvement of caspases in apoptosis. A caspase-9 inhibitor, Z-LEHD-FMK, also reduced t-BHP-induced apoptosis, suggesting that caspase-9 plays a critical role in this process. Procaspase-9 underwent cleavage in mitochondria and translocation to the nucleus, where increased caspase-9 activity was detected. The caspase-9 substrates, caspase-3 and caspase-7, were not activated. Caspase-7 was translocated from the cytosol to the endoplasmic reticulum (ER), where it underwent processing; however, enzymatic activity of caspase-7 was inhibited by t-BHP. t-BHP caused cleavage of procaspase-12 at the ER and its subsequent translocation to the nucleus, where increased caspase-12 activity was found. t-BHP caused translocation of calpain from the cytosol to the ER. Calpain inhibition reduced chromatin condensation and caspase-12 activity in the nucleus, suggesting that calpain is involved in caspase-12 activation and apoptosis. This study demonstrates that caspase-9 and caspase-12 are activated in t-BHP-induced apoptosis in hepatocytes. We highlight the importance of subcellular compartments such as mitochondria, ER and nuclei in the apoptotic process.

  3. Implication of caspases and subcellular compartments in tert-butylhydroperoxide induced apoptosis

    SciTech Connect

    Haidara, Khadidja; Marion, Michel; Gascon-Barre, Marielle; Denizeau, Francine; Averill-Bates, Diana A.

    2008-05-15

    Oxidative stress has been implicated in many physiopathologies including neurodegenerative diseases, cancer, cardiovascular and respiratory diseases, and in mechanisms of action of environmental toxicants. tert-butylhydroperoxide (t-BHP) is an organic lipid hydroperoxide analogue, which is commonly used as a pro-oxidant for evaluating mechanisms involving oxidative stress in cells and tissues. This study investigates mechanisms of apoptosis induced by oxidative stress in hepatocytes, in particular, the involvement of caspases and subcellular compartments. Freshly isolated hepatocytes were exposed to 0.4 mM t-BHP during 1 h. A general caspase inhibitor, Boc-D-FMK, reduced t-BHP-induced apoptosis (chromatin condensation), confirming the involvement of caspases in apoptosis. A caspase-9 inhibitor, Z-LEHD-FMK, also reduced t-BHP-induced apoptosis, suggesting that caspase-9 plays a critical role in this process. Procaspase-9 underwent cleavage in mitochondria and translocation to the nucleus, where increased caspase-9 activity was detected. The caspase-9 substrates, caspase-3 and caspase-7, were not activated. Caspase-7 was translocated from the cytosol to the endoplasmic reticulum (ER), where it underwent processing; however, enzymatic activity of caspase-7 was inhibited by t-BHP. t-BHP caused cleavage of procaspase-12 at the ER and its subsequent translocation to the nucleus, where increased caspase-12 activity was found. t-BHP caused translocation of calpain from the cytosol to the ER. Calpain inhibition reduced chromatin condensation and caspase-12 activity in the nucleus, suggesting that calpain is involved in caspase-12 activation and apoptosis. This study demonstrates that caspase-9 and caspase-12 are activated in t-BHP-induced apoptosis in hepatocytes. We highlight the importance of subcellular compartments such as mitochondria, ER and nuclei in the apoptotic process.

  4. Caspase-8: not so silently deadly

    PubMed Central

    Feltham, Rebecca; Vince, James E; Lawlor, Kate E

    2017-01-01

    Apoptosis is a caspase-dependent programmed form of cell death, which is commonly believed to be an immunologically silent process, required for mammalian development and maintenance of cellular homoeostasis. In contrast, lytic forms of cell death, such as RIPK3- and MLKL-driven necroptosis, and caspase-1/11-dependent pyroptosis, are postulated to be inflammatory via the release of damage associated molecular patterns (DAMPs). Recently, the function of apoptotic caspase-8 has been extended to the negative regulation of necroptosis, the cleavage of inflammatory interleukin-1β (IL-1β) to its mature bioactive form, either directly or via the NLRP3 inflammasome, and the regulation of cytokine transcriptional responses. In view of these recent advances, human autoinflammatory diseases that are caused by mutations in cell death regulatory machinery are now associated with inappropriate inflammasome activation. In this review, we discuss the emerging crosstalk between cell death and innate immune cell inflammatory signalling, particularly focusing on novel non-apoptotic functions of caspase-8. We also highlight the growing number of autoinflammatory diseases that are associated with enhanced inflammasome function. PMID:28197335

  5. Cleavage of the Junin Virus Nucleoprotein Serves a Decoy Function To Inhibit the Induction of Apoptosis during Infection

    PubMed Central

    Wolff, Svenja; Groseth, Allison

    2013-01-01

    The regulation of apoptosis during infection is an important factor for host survival and, in some cases, also for the virus life cycle. At the same time, mechanisms to prevent the induction of apoptosis have been observed in numerous viral pathogens, but until now the role of apoptosis during arenavirus infection has not been investigated. Junin virus (JUNV) belongs to the New World arenavirus serogroup of the Arenaviridae and is the causative agent of Argentine hemorrhagic fever. We have demonstrated that infection with JUNV in cell culture does not induce apoptosis but leads to cleavage of the nucleoprotein (NP) into discrete products resembling caspase cleavage events. Similar specific NP degradation patterns were also observed in NP-transfected cell lines, and a closer examination of the sequence of NP showed several putative caspase cleavage motifs. Point mutations that abolished these cleavage motifs were consistent with the loss of certain cleavage products. Consistent with these data, further studies showed that treatment with a caspase inhibitor also reduced NP cleavage, indicating that the observed cleavage events were occurring as a result of caspase activity with NP as a substrate. Finally, we showed that expression of NP suppresses the cleavage of caspase 3 in cells treated with an apoptosis activator. Based on these findings, we propose that NP functions as a decoy substrate for caspase cleavage in order to inhibit the induction of apoptosis in JUNV-infected cells. PMID:23077297

  6. Identification of cleavage of NS5A of C-strain classical swine fever virus.

    PubMed

    Xie, Jinxin; Guo, Huancheng; Gong, Wenjie; Jiang, Daliang; Zhang, Li; Jia, Junjie; Tu, Changchun

    2017-02-01

    NS5A is a multifunctional non-structural protein of classical swine fever virus (CSFV) that plays an important role in viral replication, but how it exerts its functions is unknown. Here, we report the cleavage of NS5A of the vaccine C-strain, resulting in two truncated forms (b and c). Further experiments using calpain- and caspase-family-specific inhibitors, followed by a caspase-6-specific shRNAs and inhibitor, showed that the cleavage of C-strain NS5A to produce truncated form c is mediated by caspase-6, mapping to (272)DTTD(275), while the cleavage producing truncated form b is probably mediated by another unknown protease. shRNA-mediated downregulation of caspase-6 and blocking of enzyme activity in ST cells significantly impaired genome replication and virus production, indicating that NS5A cleavage is required for CSFV replication.

  7. SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda.

    PubMed

    Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J; Clem, Rollie J

    2013-05-01

    Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    SciTech Connect

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce rRNA cleavage via

  9. Caspase-2 is involved in cell death induction by taxanes in breast cancer cells

    PubMed Central

    2013-01-01

    Background We studied the role of caspase-2 in apoptosis induction by taxanes (paclitaxel, novel taxane SB-T-1216) in breast cancer cells using SK-BR-3 (nonfunctional p53, functional caspase-3) and MCF-7 (functional p53, nonfunctional caspase-3) cell lines. Results Both taxanes induced apoptosis in SK-BR-3 as well as MCF-7 cells. Caspase-2 activity in SK-BR-3 cells increased approximately 15-fold within 48 h after the application of both taxanes at the death-inducing concentration (100 nM). In MCF-7 cells, caspase-2 activity increased approximately 11-fold within 60 h after the application of taxanes (300 nM). Caspase-2 activation was confirmed by decreasing levels of procaspase-2, increasing levels of cleaved caspase-2 and the cleavage of caspase-2 substrate golgin-160. The inhibition of caspase-2 expression using siRNA increased the number of surviving cells more than 2-fold in MCF-7 cells, and at least 4-fold in SK-BR-3 cells, 96 h after the application of death-inducing concentration of taxanes. The inhibition of caspase-2 expression also resulted in decreased cleavage of initiator caspases (caspase-8, caspase-9) as well as executioner caspases (caspase-3, caspase-7) in both cell lines after the application of taxanes. In control cells, caspase-2 seemed to be mainly localized in the nucleus. After the application of taxanes, it was released from the nucleus to the cytosol, due to the long-term disintegration of the nuclear envelope, in both cell lines. Taxane application led to some formation of PIDDosome complex in both cell lines within 24 h after the application. After taxane application, p21WAF1/CIP1 expression was only induced in MCF-7 cells with functional p53. However, taxane application did not result in a significant increase of PIDD expression in either SK-BR-3 or MCF-7 cells. The inhibition of RAIDD expression using siRNA did not affect the number of surviving SK-BR-3 and MCF-7 cells after taxane application at all. Conclusion Caspase-2 is

  10. Helicobacter pylori inhibits the cleavage of TRAF1 via a CagA-dependent mechanism.

    PubMed

    Wan, Xiu-Kun; Yuan, Sheng-Ling; Wang, Yan-Chun; Tao, Hao-Xia; Jiang, Wei; Guan, Zhang-Yan; Cao, Cheng; Liu, Chun-Jie

    2016-12-28

    To study the impact on cleavage of tumor necrosis factor receptor-associated factor 1 (TRAF1) regulated by Helicobacter pylori (H. pylori). Cleavage of TRAF1 was detected by western blotting in the human gastric cancer cell line AGS following treatment with an apoptosis inducer. Cleavage of TRAF1 mediated by caspase was examined in vitro using specific caspase inhibitors. The effect of the COOH-terminal TRAF1 fragment on gastric cell apoptosis during H. pylori infection was measured using flow cytometry. The impact of H. pylori infection on TRAF1 cleavage was detected in the presence of apoptosis inducer. The roles of H. pylori virulence factors that may regulate TRAF1 cleavage were analyzed using isogenic cagA-, vacA- and cagE-null mutants. TRAF1 was found to be cleaved in AGS cells treated with the apoptosis inducer, and caspase-8 was the major caspase involved in the cleavage of TRAF1. The COOH-terminal TRAF1 fragment significantly induced cell apoptosis (P < 0.05) as well as promoted H. pylori-induced cell apoptosis (P < 0.05). H. pylori infection was found to significantly inhibit the cleavage of TRAF1 and to inhibit the activation of caspase-8 in the presence of the apoptosis inducer at specific infection times and different cell/bacteria ratios. We also found that the effects of cagE- and cagA-null mutants on the inhibition of TRAF1 cleavage and activation of caspase-8 were significantly attenuated, compared with wild-type H. pylori, in the presence of the apoptosis inducer, showing that the virulence factor CagA was mainly involved in the inhibition of TRAF1 cleavage. H. pylori infection significantly inhibits the cleavage of TRAF1 via a CagA-dependent mechanism, which would increase the relative amounts of full-length TRAF1 and exert an antiapoptotic effect on H. pylori-infected cells.

  11. Activation of specific apoptotic caspases with an engineered small-molecule-activated protease.

    PubMed

    Gray, Daniel C; Mahrus, Sami; Wells, James A

    2010-08-20

    Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.

  12. Activation of caspase-3 in permanent and transient brain ischaemia in man.

    PubMed

    Love, S; Barber, R; Srinivasan, A; Wilcock, G K

    2000-08-03

    Animal studies have shown brain ischaemia to cause oxidative damage to DNA and activation of caspase-3, leading to apoptosis. These changes may be exacerbated by reperfusion. To assess caspase-3 activation after transient and permanent brain ischaemia in man, we examined brain tissue from patients who had experienced a cardiac arrest with resuscitation or an atherothrombotic brain infarct, and died 12 h to 9 days later. Sections were immunostained for activated caspase-3 or the 89 kDa caspase-3-mediated cleavage product of poly(ADP-ribose) polymerase. Brain ischaemia caused activation of caspase-3 in macrophages/microglia. Some neurons showed delayed activation of caspase-3 after cardiac arrest, but very few in atherothrombotic infarcts. In man, activation of caspase-3 plays little part in neuronal death in atherothrombotic infarcts but may contribute to delayed death of neurons after cardiac arrest.

  13. A novel Death Defying Domain in Met entraps the active site of Caspase-3 and blocks apoptosis in hepatocytes

    PubMed Central

    Ma, Jihong; Zou, Chunbin; Guo, Lida; Seneviratne, Danushka S.; Tan, Xinping; Kwon, Yong-Kook; An, Jiyan; Bowser, Robert; DeFrances, Marie C.; Zarnegar, Reza

    2013-01-01

    Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF) is known to function as a potent anti-apoptotic mediator in normal and neoplastic cells. Herein we report that intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of Caspase-3 cleavage sites, which bait, trap and disable the active site of Caspase-3, thereby blocking the execution of apoptosis. We call this Caspase-3 cleavage motif the ‘Death Defying Domain’ (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by Caspase-3, the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of Caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of Caspase-3 activity. By gain and loss-of-function studies using restoration of DDD expression in DDD deficient hepatocytic cells, we found that both Caspase-3 sites in DDD are necessary for inhibition of Caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met’s enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of Caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion Our findings show that Met can directly inhibit Caspase-3 via a novel mechanism and promote hepato-cyte survival. Results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play. PMID:24122846

  14. Caspase activation inhibits proteasome function during apoptosis.

    PubMed

    Sun, Xiao-Ming; Butterworth, Michael; MacFarlane, Marion; Dubiel, Wolfgang; Ciechanover, Aaron; Cohen, Gerald M

    2004-04-09

    The ubiquitin/proteasome system regulates protein turnover by degrading polyubiquitinated proteins. To date, all studies on the relationship of apoptosis and the proteasome have emphasized the key role of the proteasome in the regulation of apoptosis, by virtue of its ability to degrade regulatory molecules involved in apoptosis. We now demonstrate how induction of apoptosis may regulate the activity of the proteasome. During apoptosis, caspase activation results in the cleavage of three specific subunits of the 19S regulatory complex of the proteasome: S6' (Rpt5) and S5a (Rpn10), whose role is to recognize polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a and S2 (Rpn1) holds together the lid and base of the 19S regulatory complex. This caspase-mediated cleavage inhibits the proteasomal degradation of ubiquitin-dependent and -independent cellular substrates, including proapoptotic molecules such as Smac, so facilitating the execution of the apoptotic program by providing a feed-forward amplification loop.

  15. Activation of caspase-3 in HL-60 cells treated with pyrithione and zinc.

    PubMed

    Kondoh, Masuo; Tasaki, Emi; Takiguchi, Masufumi; Higashimoto, Minoru; Watanabe, Yoshiteru; Sato, Masao

    2005-04-01

    The transition metal zinc (Zn) is an endogenous regulator of apoptosis. The ability of Zn to modulate apoptosis is believed to be mediated by the regulation of caspase activity. Previously, we reported that an acute influx of labile Zn induced apoptosis via activation of caspase in human leukemia HL-60 cells treated with a Zn ionophore (Py, pyrithione) and Zn at 1 and 25 microM, respectively. In the present study, we investigated the involvement of caspase-3 in Py (1 microM)/Zn (25 microM)-induced apoptosis in HL-60 cells. Pro-caspase-3 is an inactive form of caspase-3. The processing of pro-caspase-3, a sign of caspase-3 activation, occurred 6 h after treatment with Py/Zn. Proteolysis of poly (ADP-ribose) polymerase (PARP), a substrate of caspase-3, was also observed 6 h after treatment with Py/Zn. We also confirmed the elevation of caspase-3 activity as an index of the cleavage of amino acid sequences recognized by activated caspase-3. An inhibitor of caspase-3 attenuated the appearance of the DNA ladder. Taken together, these results indicate that the activation of caspase-3 is partly responsible for the induction of apoptosis in Py/Zn-treated HL-60 cells.

  16. The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities

    PubMed Central

    Wu, Chu-Chiao; Lee, Sunhee; Malladi, Srinivas; Chen, Miao-Der; Mastrandrea, Nicholas J.; Zhang, Zhiwen; Bratton, Shawn B.

    2016-01-01

    According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function. PMID:27882936

  17. A fluorescent reporter of caspase activity for live imaging

    PubMed Central

    Bardet, Pierre-Luc; Kolahgar, Golnar; Mynett, Anita; Miguel-Aliaga, Irene; Briscoe, James; Meier, Pascal; Vincent, Jean-Paul

    2008-01-01

    There is a growing interest in the mechanisms that control the apoptosis cascade during development and adult life. To investigate the regulatory events that trigger apoptosis in whole tissues, we have devised a genetically encoded caspase sensor that can be detected in live and fixed tissue by standard confocal microscopy. The sensor comprises two fluorophores, mRFP, monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP), that are linked by an efficient and specific caspase-sensitive site. Upon caspase activation, the sensor is cleaved and eGFP translocates to the nucleus, leaving mRFP at membranes. This is detected before other markers of apoptosis, including anti–cleaved caspase 3 immunoreactivity. Moreover, the sensor does not perturb normal developmental apoptosis and is specific, as cleavage does not occur in Drosophila embryos that are unable to activate the apoptotic cascade. Importantly, dying cells can be recognized in live embryos, thus opening the way for in vivo imaging. As expected from the high conservation of caspases, it is also cleaved in dying cells of chick embryos. It is therefore likely to be generally useful to track the spatiotemporal pattern of caspase activity in a variety of species. PMID:18779587

  18. ATM kinase promotes both caspase-8 and caspase-9 activation during TNF-α-induced apoptosis of HeLa cells.

    PubMed

    Liu, Linhua; Yim, Hyungshin; Choi, Jae Hyuk; Kim, Seung-Tak; Jin, Yinghua; Lee, Seung Ki

    2014-03-18

    In this study, we show that atraxia telangiectasia mutated kinase (ATM) activity is generally upregulated by different apoptotic stimuli, i.e. TNF-α, TRAIL, paclitaxel, or UV. Apoptotic progression is markedly attenuated by siATM-RNA through down regulation of caspase-8 and caspase-9 in parallel with decreases in FLIP-S (short form of cellular FLICE inhibitory protein) protein levels and Bid cleavage. In addition, ATM activity is upregulated through t-Cdc6 while caspase-8 and caspase-9 activities increase. Taken together, we suggest that ATM regulates caspase-8 activation by influencing levels of FLIP-S, ATM kinase activity is upregulated by t-Cdc6, and increased ATM activity plays an essential role in the amplification of apoptosis in TNF-α-stimulated HeLa cells.

  19. Caspase-1 and IL-1β processing in a teleost fish.

    PubMed

    Reis, Marta I R; do Vale, Ana; Pereira, Pedro J B; Azevedo, Jorge E; Dos Santos, Nuno M S

    2012-01-01

    Interleukine-1β (IL-1β) is the most studied pro-inflammatory cytokine, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges. In mammals, IL-1β is synthesized as an inactive 31 kDa precursor that is cleaved by caspase-1 generating a 17.5 kDa secreted active mature form. The caspase-1 cleavage site strictly conserved in all mammalian IL-1β sequences is absent in IL-1β sequences reported for non-mammalian vertebrates. Recently, fish caspase-1 orthologues have been identified in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) but very little is known regarding their processing and activity. In this work it is shown that sea bass caspase-1 auto-processing is similar to that of the human enzyme, resulting in active p24/p10 and p20/p10 heterodimers. Moreover, the presence of alternatively spliced variants of caspase-1 in sea bass is reported. The existence of caspase-1 isoforms in fish and in mammals suggests that they have been evolutionarily maintained and therefore are likely to play a regulatory role in the inflammatory response, as shown for other caspases. Finally, it is shown that sea bass and avian IL-1β are specifically cleaved by caspase-1 at different but phylogenetically conserved aspartates, distinct from the cleavage site of mammalian IL-1β.

  20. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow; Mary Ann D.; Dahlberg, James E.

    2010-11-09

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  1. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor L.; Brow, Mary Ann D.; Dahlberg, James E.

    2007-12-11

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  2. Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation.

    PubMed

    Mead, Emma; Kestoras, Dimitra; Gibson, Yolanda; Hamilton, Lucy; Goodson, Ross; Jones, Sophie; Eversden, Sarah; Davies, Peter; O'Neill, Michael; Hutton, Michael; Szekeres, Philip; Wolak, Joanna

    2016-10-18

    Intracellular neurofibrillary tangles (NFTs) are the hallmark of Alzheimer's disease and other tauopathies in which tau, a microtubule-associated protein, loses its ability to stabilize microtubules. Several post-translational modifications including phosphorylation and truncation increase tau's propensity to aggregate thus forming NFTs; however, the mechanisms underlying tau conformational change and aggregation still remain to be defined. Caspase activation and subsequent proteolytic cleavage of tau is thought to be a potential trigger of this disease-related pathological conformation. The aim of this work was to investigate the link between caspase activation and a disease-related conformational change of tau in a neuroblastoma cell-based model of spontaneous tau aggregation. We demonstrated that caspase induction initiates proteolytic cleavage of tau and generation of conformationally altered and aggregated tau recognized by the MC1 conformational antibody. Most importantly, these events were shown to be attenuated with caspase inhibitors. This implies that therapeutics aimed at inhibiting caspase-mediated tau cleavage may prove beneficial in slowing cleavage and aggregation, thus potentially halting tau pathology and disease progression.

  3. Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

    PubMed Central

    Mead, Emma; Kestoras, Dimitra; Gibson, Yolanda; Hamilton, Lucy; Goodson, Ross; Jones, Sophie; Eversden, Sarah; Davies, Peter; O’Neill, Michael; Hutton, Michael; Szekeres, Philip; Wolak, Joanna

    2016-01-01

    Intracellular neurofibrillary tangles (NFTs) are the hallmark of Alzheimer’s disease and other tauopathies in which tau, a microtubule-associated protein, loses its ability to stabilize microtubules. Several post-translational modifications including phosphorylation and truncation increase tau’s propensity to aggregate thus forming NFTs; however, the mechanisms underlying tau conformational change and aggregation still remain to be defined. Caspase activation and subsequent proteolytic cleavage of tau is thought to be a potential trigger of this disease-related pathological conformation. The aim of this work was to investigate the link between caspase activation and a disease-related conformational change of tau in a neuroblastoma cell-based model of spontaneous tau aggregation. We demonstrated that caspase induction initiates proteolytic cleavage of tau and generation of conformationally altered and aggregated tau recognized by the MC1 conformational antibody. Most importantly, these events were shown to be attenuated with caspase inhibitors. This implies that therapeutics aimed at inhibiting caspase-mediated tau cleavage may prove beneficial in slowing cleavage and aggregation, thus potentially halting tau pathology and disease progression. PMID:27589517

  4. Roles of inflammatory caspases during processing of zebrafish interleukin-1β in Francisella noatunensis infection

    USGS Publications Warehouse

    Vojtech, Lucia N.; Scharping, Nichole; Woodson, James C.; Hansen, John D.

    2012-01-01

    The interleukin-1 family of cytokines are essential for the control of pathogenic microbes but are also responsible for devastating autoimmune pathologies. Consequently, tight regulation of inflammatory processes is essential for maintaining homeostasis. In mammals, interleukin-1 beta (IL-1β) is primarily regulated at two levels, transcription and processing. The main pathway for processing IL-1β is the inflammasome, a multiprotein complex that forms in the cytosol and which results in the activation of inflammatory caspase (caspase 1) and the subsequent cleavage and secretion of active IL-1β. Although zebrafish encode orthologs of IL-1β and inflammatory caspases, the processing of IL-1β by activated caspase(s) has never been examined. Here, we demonstrate that in response to infection with the fish-specific bacterial pathogen Francisella noatunensis, primary leukocytes from adult zebrafish display caspase-1-like activity that results in IL-1β processing. Addition of caspase 1 or pancaspase inhibitors considerably abrogates IL-1β processing. As in mammals, this processing event is concurrent with the secretion of cleaved IL-1β into the culture medium. Furthermore, two putative zebrafish inflammatory caspase orthologs, caspase A and caspase B, are both able to cleave IL-1β, but with different specificities. These results represent the first demonstration of processing and secretion of zebrafish IL-1β in response to a pathogen, contributing to our understanding of the evolutionary processes governing the regulation of inflammation.                   

  5. CASPASE-8 gene is inactivated by somatic mutations in gastric carcinomas.

    PubMed

    Soung, Young Hwa; Lee, Jong Woo; Kim, Su Young; Jang, Jin; Park, Yong Gyu; Park, Won Sang; Nam, Suk Woo; Lee, Jung Young; Yoo, Nam Jin; Lee, Sug Hyung

    2005-02-01

    Several lines of evidence indicate that deregulation of apoptosis is involved in the mechanisms of cancer development. Caspase-8 activation plays a central role in the initiation phase of apoptosis. The aim of this study was to explore the possibility that genetic alteration of CASPASE-8 gene is involved in the development of human cancers, including gastric cancers. We have analyzed the entire coding region of human CASPASE-8 gene for the detection of somatic mutations in 162 gastric carcinomas (40 early and 122 advanced cancers), 185 non-small cell lung cancers, 93 breast carcinomas, and 88 acute leukemias by PCR-single-strand conformation polymorphism. Of the cancers analyzed, 13 cancers harbored CASPASE-8 somatic mutations. Interestingly, all of the mutations were detected in the advanced gastric cancers (10.7% of the 122 samples). We expressed the tumor-derived caspase-8 mutants in 293T, 293, and HT1080 cells and found that most of the mutants (9 of the 10 mutations tested) markedly decreased the cell death activity of caspase-8. In addition, in the cells with the inactivating caspase-8 mutants, cleavage of poly(ADP-ribose)polymerase was markedly reduced compared with that of wild-type caspase-8. The occurrence of CASPASE-8 mutation and the inactivation of cell death activity by the mutants suggest that CASPASE-8 gene mutation may affect the pathogenesis of gastric cancers, especially at the late stage of gastric carcinogenesis.

  6. Revisiting caspases in sepsis

    PubMed Central

    Aziz, M; Jacob, A; Wang, P

    2014-01-01

    Sepsis is a life-threatening illness that occurs due to an abnormal host immune network which extends through the initial widespread and overwhelming inflammation, and culminates at the late stage of immunosupression. Recently, interest has been shifted toward therapies aimed at reversing the accompanying periods of immune suppression. Studies in experimental animals and critically ill patients have demonstrated that increased apoptosis of lymphoid organs and some parenchymal tissues contributes to this immune suppression, anergy and organ dysfunction. Immediate to the discoveries of the intracellular proteases, caspases for the induction of apoptosis and inflammation, and their striking roles in sepsis have been focused elaborately in a number of original and review articles. Here we revisited the different aspects of caspases in terms of apoptosis, pyroptosis, necroptosis and inflammation and focused their links in sepsis by reviewing several recent findings. In addition, we have documented striking perspectives which not only rewrite the pathophysiology, but also modernize our understanding for developing novel therapeutics against sepsis. PMID:25412304

  7. Targets and Intracellular Signaling Mechanisms for Deoxynivalenol-Induced Ribosomal RNA Cleavage

    PubMed Central

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-01-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. PMID:22491426

  8. Targets and intracellular signaling mechanisms for deoxynivalenol-induced ribosomal RNA cleavage.

    PubMed

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    2012-06-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3.

  9. ARTEMIS nuclease facilitates apoptotic chromatin cleavage.

    PubMed

    Britton, Sébastien; Frit, Philippe; Biard, Denis; Salles, Bernard; Calsou, Patrick

    2009-10-15

    One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis.

  10. Identification and Evaluation of Novel Small Molecule Pan-Caspase Inhibitors in Huntington’s Disease Models

    PubMed Central

    Leyva, Melissa J.; DeGiacomo, Francesco; Kaltenbach, Linda S.; Holcomb, Jennifer; Zhang, Ningzhe; Gafni, Juliette; Park, Hyunsun; Lo, Donald C.; Salvesen, Guy S.; Ellerby, Lisa M.; Ellman, Jonathan A.

    2010-01-01

    SUMMARY Huntington’s Disease (HD) is characterized by a mutation in the huntingtin gene encoding an expansion of glutamine repeats on the N-terminus of the huntingtin (Htt) protein. Numerous studies have identified Htt proteolysis as a critical pathological event in post mortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD targets. We report the use of the substrate activity screening method against caspases-3 and -6 to identify three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In HD models, these irreversible inhibitors suppressed Hdh111Q/111Q-mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study the identified nonpeptidic caspase inhibitors were used to confirm the role of caspase-mediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development. PMID:21095569

  11. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles.

    PubMed

    Julien, Olivier; Zhuang, Min; Wiita, Arun P; O'Donoghue, Anthony J; Knudsen, Giselle M; Craik, Charles S; Wells, James A

    2016-04-05

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

  12. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

    PubMed Central

    Zhuang, Min; Wiita, Arun P.; O’Donoghue, Anthony J.; Knudsen, Giselle M.; Craik, Charles S.; Wells, James A.

    2016-01-01

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events. PMID:27006500

  13. Blockade of processing/activation of caspase-3 by hypoxia

    SciTech Connect

    Han, Sang Hee; Kim, Moonil; Park, Kyoungsook; Kim, Tae-Hyoung; Seol, Dai-Wu

    2008-10-31

    Tumor hypoxia, which is caused by the rapid proliferation of tumor cells and aberrant vasculature in tumors, results in inadequate supplies of oxygen and nutrients to tumor cells. Paradoxically, these unfavorable growth conditions benefit tumor cell survival, although the mechanism is poorly understood. We have demonstrated for the first time that hypoxia inhibits TRAIL-induced apoptosis by blocking translocation of Bax from cytosol to the mitochondria in tumor cells. However, it is largely unknown how hypoxia-inhibited Bax translocation attenuates TRAIL-induced apoptosis. Here, we demonstrate that despite its inhibitory activity in TRAIL-induced apoptosis, hypoxia does not affect TRAIL-triggered proximal apoptotic signaling events, including caspase-8 activation and Bid cleavage. Instead, hypoxia inhibited processing of caspase-3, leading to incomplete activation of the caspase. Importantly, hypoxia-blocked translocation of Bax to the mitochondria significantly inhibited releasing the mitochondrial factors, such as cytochrome c and Smac/DIABLO, to the cytosol in response to TRAIL. It is well-known that complete processing/activation of caspase-3 requires Smac/DIABLO released from mitochondria. Therefore, our data indicate that an engagement of the apoptotic mitochondrial events leading to caspase-3 activation is blocked by hypoxia. Our data shed new light on understanding of the apoptotic signal transduction and targets regulated by tumor hypoxia.

  14. Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death.

    PubMed

    Chandra, Dhyan; Choy, Grace; Deng, Xiaodi; Bhatia, Bobby; Daniel, Peter; Tang, Dean G

    2004-08-01

    It was recently demonstrated that during apoptosis, active caspase 9 and caspase 3 rapidly accumulate in the mitochondrion-enriched membrane fraction (D. Chandra and D. G. Tang, J. Biol. Chem.278:17408-17420, 2003). We now show that active caspase 8 also becomes associated with the membranes in apoptosis caused by multiple stimuli. In MDA-MB231 breast cancer cells treated with etoposide (VP16), active caspase 8 is detected only in the membrane fraction, which contains both mitochondria and endoplasmic reticulum (ER), as revealed by fractionation studies. Immunofluorescence microscopy, however, shows that procaspase 8 and active caspase 8 predominantly colocalize with the mitochondria. Biochemical analysis demonstrates that both procaspase 8 and active caspase 8 are localized mainly on the outer mitochondrial membrane (OMM) as integral proteins. Functional analyses with dominant-negative mutants, small interfering RNAs, peptide inhibitors, and Fas-associated death domain (FADD)- and caspase 8-deficient Jurkat T cells establish that the mitochondrion-localized active caspase 8 results mainly from the FADD-dependent and tumor necrosis factor receptor-associated death domain-dependent mechanisms and that caspase 8 activation plays a causal role in VP16-induced caspase 3 activation and cell death. Finally, we present evidence that the OMM-localized active caspase 8 can activate cytosolic caspase 3 and ER-localized BAP31. Cleavage of BAP31 leads to the generation of ER- localized, proapoptotic BAP20, which may mediate mitochondrion-ER cross talk through a Ca(2+)-dependent mechanism.

  15. A new colorimetric strategy for monitoring caspase 3 activity by HRP-mimicking DNAzyme-peptide conjugates.

    PubMed

    Zhou, Zhaojuan; Peng, Lu; Wang, Xiaoyan; Xiang, Yu; Tong, Aijun

    2014-03-07

    A new method for caspase 3 activity assay has been developed based on HRP-mimicking DNAzyme-peptide conjugates. The mechanism of detection was based on the specific cleavage of DEVD-peptides by active caspase 3 for recognition and the catalytic properties of HRP-mimicking DNAzymes for signal amplification. Under optimal conditions, the detection limit of caspase 3 was 0.89 nM. The proposed method was also successfully applied for the detection of caspase 3 in apoptosis cell lysates.

  16. Galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-Bid mitochondrial pathway.

    PubMed

    Zhang, Hai-Tao; Wu, Jun; Wen, Min; Su, Li-Juan; Luo, Hui

    2012-01-01

    This study has investigated whether galangin, a flavonol derived from Alpinia officinarum Hance and used as food additives in southern China, induces apoptosis in hepatocellular carcinoma cells (HCCs) by activation of the caspase-8 and Bid pathway. The apoptosis of HCCs was evaluated by in situ uptake of propidium iodide and Hoechst 33258. Protein expressions were detected by Western blotting. Caspase-8 activity was measured using colorimetric method. To confirm the galangin-induced apoptotic pathway, inhibition of caspase-8 activity by Z-IETD-FMK, knockdown of Bid expression with siRNA, and overexpression of Bcl-2 in cells were carried out, respectively. The results show that galangin has significantly induced apoptosis in HCC lines. The caspase-8 is activated, and the cleavage of Bid results in the increase in tBid. The galangin-induced apoptosis is attenuated by Z-IETD-FMK, Bid siRNA, and Bcl-2 overexpression, respectively. However, Bcl-2 fails to suppress caspase-8 activation and the cleavage of Bid. This study has demonstrated that galangin induces apoptosis in HCCs by activating caspase 8/t-Bid mitochondrial pathway. Although Bcl-2 overexpression attenuates galangin-mediated apoptosis of HCCs, it is not mediated by the inhibition of tBid generation and caspase-8 activation.

  17. Coordination between proteasome impairment and caspase activation leading to TAU pathology: neuroprotection by cAMP

    PubMed Central

    Metcalfe, M J; Huang, Q; Figueiredo-Pereira, M E

    2012-01-01

    Neurofibrillary tangles (NFTs) are hallmarks of Alzheimer's disease (AD). The main component of NFTs is TAU, a highly soluble microtubule-associated protein. However, when TAU is cleaved at Asp421 by caspases it becomes prone to aggregation leading to NFTs. What triggers caspase activation resulting in TAU cleavage remains unclear. We investigated in rat cortical neurons a potential coordination between proteasome impairment and caspase activation. We demonstrate that upon proteasome inhibition, the early accumulation of detergent-soluble ubiquitinated (SUb) proteins paves the way to caspase activation and TAU pathology. This occurs with two drugs that inhibit the proteasome by different means: the product of inflammation prostaglandin J2 (PGJ2) and epoxomicin. Our results pinpoint a critical early event, that is, the buildup of SUb proteins that contributes to caspase activation, TAU cleavage, TAU/Ub-protein aggregation and neuronal death. Furthermore, to our knowledge, we are the first to demonstrate that elevating cAMP in neurons with dibutyryl-cAMP (db-cAMP) or the lipophilic peptide PACAP27 prevents/diminishes caspase activation, TAU cleavage and neuronal death induced by PGJ2, as long as these PGJ2-induced changes are moderate. db-cAMP also stimulated proteasomes, and mitigated proteasome inhibition induced by PGJ2. We propose that targeting cAMP/PKA to boost proteasome activity in a sustainable manner could offer an effective approach to avoid early accumulation of SUb proteins and later caspase activation, and TAU cleavage, possibly preventing/delaying AD neurodegeneration. PMID:22717581

  18. Targeting caspases in cancer therapeutics

    PubMed Central

    Hensley, Patrick; Mishra, Murli; Kyprianou, Natasha

    2013-01-01

    The identification of the fundamental role of apoptosis in the growth balance and normal homeostasis against cell proliferation led to the recognition of its loss contributing to tumorigenesis. The mechanistic significance of reinstating apoptosis signaling towards selective targeting of malignant cells heavily exploits the caspase family of death-inducing molecules as a powerful therapeutic platform for the development of potent anticancer strategies. Some apoptosis inhibitors induce caspase expression and activity in preclinical models and clinical trials by targeting both the intrinsic and extrinsic apoptotic pathways and restoring the apoptotic capacity in human tumors. Furthermore, up-regulation of caspases emerges as a sensitizing mechanism for tumors exhibiting therapeutic resistance to radiation and adjuvant chemotherapy. This review provides a comprehensive discussion of the functional involvement of caspases in apoptosis control and the current understanding of reactivating caspase-mediated apoptosis signaling towards effective therapeutic modalities in cancer treatment. PMID:23509217

  19. Specific inhibition of caspase-3 by a competitive DARPin: molecular mimicry between native and designed inhibitors.

    PubMed

    Schroeder, Thilo; Barandun, Jonas; Flütsch, Andreas; Briand, Christophe; Mittl, Peer R E; Grütter, Markus G

    2013-02-05

    Dysregulation of apoptosis is associated with several human diseases. The main apoptotic mediators are caspases, which propagate death signals to downstream targets. Executioner caspase-3 is responsible for the majority of cleavage events and its therapeutic potential is of high interest with to date several available active site peptide inhibitors. These molecules inhibit caspase-3, but also homologous caspases. Here, we describe caspase-3 specific inhibitors D3.4 and D3.8, which have been selected from a library of designed ankyrin repeat proteins (DARPins). The crystal structures of D3.4 and mutants thereof show how high specificity and inhibition is achieved. They also show similarities in the binding mode with that of the natural caspase inhibitor XIAP (X-linked inhibitor of apoptosis). The kinetic data reveal a competitive inhibition mechanism. D3.4 is specific for caspase-3 and does not bind the highly homologous caspase-7. D3.4 therefore is an excellent tool to define the precise role of caspase-3 in the various apoptotic pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function.

    PubMed

    Guey, Baptiste; Bodnar, Mélanie; Manié, Serge N; Tardivel, Aubry; Petrilli, Virginie

    2014-12-02

    Inflammasomes are caspase-1-activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11(-/-) cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.

  1. Label-Free and Homogenous Detection of Caspase-3-Like Proteases by Disrupting Homodimerization-Directed Bipartite Tetracysteine Display.

    PubMed

    Yang, Yong; Liang, Yan; Zhang, Chun-Yang

    2017-04-04

    Caspase-3-like proteases (e.g., caspase-3 and caspase-7) constitute the core players of cell apoptosis, and their dysregulation has been linked to a number of human diseases, such as cancer and neurodegenerative disorders. However, the methods for measuring caspase-3-like proteases are often complex and time-consuming. Herein, we develop a label-free method to homogeneously detect caspase-3-like proteases in vitro and in complex cell lysate. This assay uses a modular peptide that contains a dimerization domain, a caspase-3/7 cleavage sequence, and a dicysteine motif as the activity sensor to detect caspase-3-like proteases. In the absence of caspase-3-like proteases, the homodimerization of modular peptide brings the dicysteine motif into close proximity and forms a particular configuration suitable for the binding to bisarsenical dye FlAsH-EDT2. The coordination of FlAsH-EDT2 to dimeric peptide forms a highly fluorescent FlAsH-peptide complex. In contrast, the cleavage of the modular peptide by caspase-3-like proteases removes the dicysteine motif from the peptide and abrogates the bipartite tetracysteine display, leading to the disappearance of fluorescence. As a result, the caspase-3-like proteases can be quantitatively evaluated by measuring the change in fluorescence. This assay may be carried out in a "mix-and-read" manner and is, thus, quite simple and convenient. Moreover, this assay is extremely specific and can measure caspase-3 protein down to 1.28 × 10(-4) μg/mL. Importantly, the assay is compatible with complex biological samples and can measure both the activation and inhibition of intracellular caspase-3, thereby providing a new approach for the screening of caspase-targeted drugs and the diagnosis of apoptosis-associated diseases.

  2. Caspase-2: vestigial remnant or master regulator?

    PubMed

    Troy, Carol M; Ribe, Elena M

    2008-09-23

    Caspase-2, the second mammalian caspase to be identified and the most evolutionarily conserved caspase, has eluded classification. The lack of a profound phenotype in the caspase-2-deficient mouse resulted in decreased interest in caspase-2 for many years. However, advances in the field, including the identification of a potential activation complex and the development of methods to detect active caspase-2, now illuminate our understanding of the function of this caspase. These studies suggest that caspase-2 induces death through two pathways. First, caspase-2 induces cell death independently of the mitochondrial pathway, in a manner similar to that of ced-3, a caspase in Caenorhabditis elegans. Second, caspase-2 also induces cell death upstream of the mitochondrial pathway. The choice of pathway may depend on the type of death stimulus. The placing of caspase-2 upstream and independent of mitochondrial dysfunction provides a potentially new therapeutic target for aberrant cell death.

  3. Matrin 3 is a Ca{sup 2+}/calmodulin-binding protein cleaved by caspases

    SciTech Connect

    Alexander Valencia, C.; Ju, Wujian; Liu, Rihe . E-mail: rihe_liu@unc.edu

    2007-09-21

    Matrin 3 is a nuclear matrix protein that has been implicated in interacting with other nuclear proteins to anchor hyperedited RNAs to the nuclear matrix, in modulating the activity of proximal promoters, and as the main PKA substrate following NMDA receptor activation. In our proteome-wide selections for calmodulin (CaM) binding proteins and for caspase substrates using mRNA-displayed human proteome libraries, matrin 3 was identified as both a Ca{sup 2+}-dependent CaM-binding protein and a downstream substrate of caspases. We report here, the in vitro characterization of the CaM-binding motif and the caspase cleavage site on matrin 3. Significantly, the Ca{sup 2+}/CaM-binding motif is partially overlapped by the RRM of matrin 3 and is also very close to the bipartite NLS that is essential for its nuclear localization. The caspase cleavage site is downstream of the NLS but upstream of the second U1-like zinc finger. Our results suggest that the functions of matrin 3 could be regulated by both Ca{sup 2+}-dependent interaction with CaM and caspase-mediated cleavage.

  4. Developmental Truncations of Connexin 50 by Caspases Adaptively Regulate Gap Junctions/Hemichannels and Protect Lens Cells against Ultraviolet Radiation*

    PubMed Central

    Wang, Ke; Gu, Sumin; Yin, Xinye; Weintraub, Susan T.; Hua, Zichun; Jiang, Jean X.

    2012-01-01

    The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging. PMID:22418432

  5. Targeting caspases in intracellular protozoan infections.

    PubMed

    Guillermo, Landi V C; Pereira, Wânia F; De Meis, Juliana; Ribeiro-Gomes, Flavia L; Silva, Elisabeth M; Kroll-Palhares, Karina; Takiya, Christina M; Lopes, Marcela F

    2009-06-01

    Caspases are cysteine aspartases acting either as initiators (caspases 8, 9, and 10) or executioners (caspases 3, 6, and 7) to induce programmed cell death by apoptosis. Parasite infections by certain intracellular protozoans increase host cell life span by targeting caspase activation. Conversely, caspase activation, followed by apoptosis of lymphocytes and other cells, prevents effective immune responses to chronic parasite infection. Here we discuss how pharmacological inhibition of caspases might affect the immunity to protozoan infections, by either blocking or delaying apoptosis.

  6. A review of statistical methods for prediction of proteolytic cleavage.

    PubMed

    duVerle, David A; Mamitsuka, Hiroshi

    2012-05-01

    A fundamental component of systems biology, proteolytic cleavage is involved in nearly all aspects of cellular activities: from gene regulation to cell lifecycle regulation. Current sequencing technologies have made it possible to compile large amount of cleavage data and brought greater understanding of the underlying protein interactions. However, the practical impossibility to exhaustively retrieve substrate sequences through experimentation alone has long highlighted the need for efficient computational prediction methods. Such methods must be able to quickly mark substrate candidates and putative cleavage sites for further analysis. Available methods and expected reliability depend heavily on the type and complexity of proteolytic action, as well as the availability of well-labelled experimental data sets: factors varying greatly across enzyme families. For this review, we chose to give a quick overview of the general issues and challenges in cleavage prediction methods followed by a more in-depth presentation of major techniques and implementations, with a focus on two particular families of cysteine proteases: caspases and calpains. Through their respective differences in proteolytic specificity (high for caspases, broader for calpains) and data availability (much lower for calpains), we aimed to illustrate the strengths and limitations of techniques ranging from position-based matrices and decision trees to more flexible machine-learning methods such as hidden Markov models and Support Vector Machines. In addition to a technical overview for each family of algorithms, we tried to provide elements of evaluation and performance comparison across methods.

  7. Helicobacter pylori inhibits the cleavage of TRAF1 via a CagA-dependent mechanism

    PubMed Central

    Wan, Xiu-Kun; Yuan, Sheng-Ling; Wang, Yan-Chun; Tao, Hao-Xia; Jiang, Wei; Guan, Zhang-Yan; Cao, Cheng; Liu, Chun-Jie

    2016-01-01

    AIM To study the impact on cleavage of tumor necrosis factor receptor-associated factor 1 (TRAF1) regulated by Helicobacter pylori (H. pylori). METHODS Cleavage of TRAF1 was detected by western blotting in the human gastric cancer cell line AGS following treatment with an apoptosis inducer. Cleavage of TRAF1 mediated by caspase was examined in vitro using specific caspase inhibitors. The effect of the COOH-terminal TRAF1 fragment on gastric cell apoptosis during H. pylori infection was measured using flow cytometry. The impact of H. pylori infection on TRAF1 cleavage was detected in the presence of apoptosis inducer. The roles of H. pylori virulence factors that may regulate TRAF1 cleavage were analyzed using isogenic cagA-, vacA- and cagE-null mutants. RESULTS TRAF1 was found to be cleaved in AGS cells treated with the apoptosis inducer, and caspase-8 was the major caspase involved in the cleavage of TRAF1. The COOH-terminal TRAF1 fragment significantly induced cell apoptosis (P < 0.05) as well as promoted H. pylori-induced cell apoptosis (P < 0.05). H. pylori infection was found to significantly inhibit the cleavage of TRAF1 and to inhibit the activation of caspase-8 in the presence of the apoptosis inducer at specific infection times and different cell/bacteria ratios. We also found that the effects of cagE- and cagA-null mutants on the inhibition of TRAF1 cleavage and activation of caspase-8 were significantly attenuated, compared with wild-type H. pylori, in the presence of the apoptosis inducer, showing that the virulence factor CagA was mainly involved in the inhibition of TRAF1 cleavage. CONCLUSION H. pylori infection significantly inhibits the cleavage of TRAF1 via a CagA-dependent mechanism, which would increase the relative amounts of full-length TRAF1 and exert an antiapoptotic effect on H. pylori-infected cells. PMID:28082808

  8. Combined treatment with the Cox-2 inhibitor niflumic acid and PPARγ ligand ciglitazone induces ER stress/caspase-8-mediated apoptosis in human lung cancer cells.

    PubMed

    Kim, Byeong Mo; Maeng, Kyungah; Lee, Kee-Ho; Hong, Sung Hee

    2011-01-28

    The present study was performed to investigate the possible combined use of the Cox-2 inhibitor niflumic acid and the PPARγ ligand ciglitazone and to elucidate the mechanisms underlying enhanced apoptosis by this combination treatment in human lung cancer cells. Combined niflumic acid-ciglitazone treatment synergistically induced apoptotic cell death, activated caspase-9, caspase-3, and induced caspase-3-mediated PARP cleavage. The combination treatment also triggered apoptosis through caspase-8/Bid/Bax activation, and the inhibition of caspase-8 suppressed caspase-8/Bid activation, caspase-3-mediated PARP cleavage, and concomitant apoptosis. In addition, combined niflumic acid-ciglitazone treatment significantly induced ER stress responses, and suppression of CHOP expression significantly attenuated the combined niflumic acid-ciglitazone treatment-induced activation of caspase-8 and caspase-3, and the subsequent apoptotic cell death, indicating a role of ER stress in caspase-8 activation and apoptosis. Interestingly, the pro-apoptotic effects of combined niflumic acid-ciglitazone treatment were realized through Cox-2- and PPARγ-independent mechanisms. Taken together, these results suggest that sequential ER stress and caspase-8 activation are critical in combined niflumic acid-ciglitazone treatment-induced apoptosis in human lung cancer cells.

  9. Geminin is cleaved by caspase-3 during apoptosis in Xenopus egg extracts

    SciTech Connect

    Auziol, Camille; Mechali, Marcel; Maiorano, Domenico. E-Mail: maiorano@igh.cnrs.fr

    2007-09-21

    Geminin is an important cell cycle regulator having a dual role in cell proliferation and differentiation. During proliferation, Geminin controls DNA synthesis by interacting with the licensing factor Cdt1 and interferes with the onset of differentiation by inhibiting the activity of transcription factors such as Hox and Six3. During early development Geminin also functions as neural inducer. Thus differential interaction of Geminin with Cdt1 or development-specific transcription factors influence the balance between proliferation and differentiation. Here, we report an additional feature of Geminin showing that it is a novel substrate of caspase-3 during apoptosis in in vitro Xenopus egg extracts. We also show that cleavage of Geminin occurs both in solution and on chromatin with distinct kinetics. In addition we show that cleavage of Geminin by caspase-3 is not relevant to its function as regulator of DNA synthesis, suggesting that its cleavage may be relevant to its role in differentiation.

  10. Genetic ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis: the involvement of keratin-17

    PubMed Central

    Lee, Mee-Hyun; Lim, Do Young; Kim, Myoung Ok; Lee, Sung-Young; Shin, Seung Ho; Kim, Jae Young; Kim, Sung-Hyun; Kim, Dong Joon; Jung, Sung Keun; Yao, Ke; Kundu, Joydeb Kumar; Lee, Hye Suk; Lee, Cheol-Jung; Dickinson, Sally E.; Alberts, David; Bowden, G.Timothy; Stratton, Steven; Curiel, Clara; Einspahr, Janine; Bode, Ann M.; Surh, Young-Joon; Dong, Zigang

    2015-01-01

    Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17. PMID:26271098

  11. Genetic ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis: the involvement of keratin-17.

    PubMed

    Lee, Mee-Hyun; Lim, Do Young; Kim, Myoung Ok; Lee, Sung-Young; Shin, Seung Ho; Kim, Jae Young; Kim, Sung-Hyun; Kim, Dong Joon; Jung, Sung Keun; Yao, Ke; Kundu, Joydeb Kumar; Lee, Hye Suk; Lee, Cheol-Jung; Dickinson, Sally E; Alberts, David; Bowden, G Timothy; Stratton, Steven; Curiel, Clara; Einspahr, Janine; Bode, Ann M; Surh, Young-Joon; Cho, Yong-Yeon; Dong, Zigang

    2015-11-01

    Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Immunoblotting for active caspase-1.

    PubMed

    Jakobs, Christopher; Bartok, Eva; Kubarenko, Andrej; Bauernfeind, Franz; Hornung, Veit

    2013-01-01

    Immunoblotting for caspase-1 is the gold-standard method of detecting inflammasome activation. In contrast to IL-1β-based readouts, it can be used in an experimental setup independent of de novo gene expression. Here, we present protocols for the preparation and precipitation of supernatant samples containing activated caspase-1 as well as protocols for polyacrylamide gel electrophoresis (PAGE) and protein immunoblotting.

  13. Vital functions for lethal caspases.

    PubMed

    Launay, Sophie; Hermine, Olivier; Fontenay, Michaëla; Kroemer, Guido; Solary, Eric; Garrido, Carmen

    2005-08-04

    Caspases are a family of cysteine proteases expressed as inactive zymogens in virtually all animal cells. These enzymes play a central role in most cell death pathways leading to apoptosis but growing evidences implicate caspases also in nonapoptotic functions. Several of these enzymes, activated in molecular platforms referred to as inflammasomes, play a role in innate immune response by processing some of the cytokines involved in inflammatory response. Caspases are requested for terminal differentiation of specific cell types, whether this differentiation process leads to enucleation or not. These enzymes play also a role in T and B lymphocyte proliferation and, in some circumstances, appear to be cytoprotective rather than cytotoxic. These pleiotropic functions implicate caspases in the control of life and death but the fine regulation of their dual effect remains poorly understood. The nonapoptotic functions of caspases implicate that cells can restrict the proteolytic activity of these enzymes to selected substrates. Deregulation of the pathways in which caspases exert these nonapoptotic functions is suspected to play a role in the pathophysiology of several human diseases.

  14. Vps41, a protein involved in lysosomal trafficking, interacts with caspase-8.

    PubMed

    Wang, Lu; Pan, Xiao; He, Liangqiang; Zhang, Rong; Chen, Wei; Zhang, Jing; Lu, Min; Hua, Zi-Chun

    2013-01-01

    Caspase-8 is a member of the cysteine-aspartic acid protease (caspase) family which plays a central role in apoptosis and development. We screened caspase-8 interacting proteins from mouse T-cell lymphoma and 7.5-day embryo cDNA libraries by yeast two-hybrid system and obtained eleven positive clones, including Vacuolar protein sorting 41 (Vps41), a protein involved in trafficking of proteins from the late Golgi to the vacuole. The interaction of Vps41 with caspase-8 was confirmed by co-immunoprecipitation (co-IP) and co-localization studies in HEK293T cells. Co-IP experiments also showed that Vps41 binds to the p18 subunit of caspase-8 through its WD40 region and RING-finger motif. Furthermore, we found that overexpression of Vps41 promotes Fas-induced apoptosis in A549 human lung adenocarcinoma cells. The cleavage of caspase-3, a caspase-8 downstream effector, was increased when cells were transfected with Vps41-overexpressing plasmid. Together, these results suggest a novel interaction of caspase-8 with Vps41 and provide a potential role of Vps41 beyond lysosomal trafficking.

  15. Prevention of Chlamydia-Induced Infertility by Inhibition of Local Caspase Activity

    PubMed Central

    Igietseme, Joseph U.; Omosun, Yusuf; Partin, James; Goldstein, Jason; He, Qing; Joseph, Kahaliah; Ellerson, Debra; Ansari, Uzma; Eko, Francis O.; Bandea, Claudiu; Zhong, Guangming; Black, Carolyn M.

    2013-01-01

    Tubal factor infertility (TFI) represents 36% of female infertility and genital infection by Chlamydia trachomatis (C. trachomatis) is a major cause. Although TFI is associated with host inflammatory responses to bacterial components, the molecular pathogenesis of Chlamydia-induced infertility remains poorly understood. We investigated the hypothesis that activation of specific cysteine proteases, the caspases, during C. trachomatis genital infection causes the disruption of key fertility-promoting molecules required for embryo development and implantation. We analyzed the effect of caspase inhibition on infertility and the integrity of Dicer, a caspase-sensitive, fertility-promoting ribonuclease III enzyme, and key micro-RNAs in the reproductive system. Genital infection with the inflammation- and caspase-inducing, wild-type C. trachomatis serovar L2 led to infertility, but the noninflammation-inducing, plasmid-free strain did not. We confirmed that caspase-mediated apoptotic tissue destruction may contribute to chlamydial pathogenesis. Caspase-1 or -3 deficiency, or local administration of the pan caspase inhibitor, Z-VAD-FMK into normal mice protected against Chlamydia-induced infertility. Finally, the oviducts of infected infertile mice showed evidence of caspase-mediated cleavage inactivation of Dicer and alteration in critical miRNAs that regulate growth, differentiation, and development, including mir-21. These results provide new insight into the molecular pathogenesis of TFI with significant implications for new strategies for treatment and prevention of chlamydial complications. PMID:23303804

  16. Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    PubMed Central

    Perry, Mark; Granjon, Thierry; Gonzalvez, François; Gottlieb, Eyal; Ayala-Sanmartin, Jesus; Klösgen, Beate; Schwille, Petra; Petit, Patrice X.

    2013-01-01

    Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria. PMID:23418437

  17. Molecular cloning and characterisation of sea bass (Dicentrarchus labrax L.) caspase-3 gene.

    PubMed

    Reis, Marta I R; Nascimento, Diana S; do Vale, Ana; Silva, Manuel T; dos Santos, Nuno M S

    2007-02-01

    Caspase-3 is one of the major caspases operating in apoptosis, cleaving and inactivating a number of molecules and largely contributing to the apoptotic phenotype and the dismantling of the apoptoting cell. The opening reading frame of sea bass (Dicentrarchus labrax L.) caspase-3 has 281 amino acids. The complete sequence of caspase-3 shows a very close homology to the correspondent sequence from other vertebrates, in particularly with that of Takifugu rubripes and Oryzias latipes, with 87.7 and 87.9% of similarity, respectively. Furthermore, the sea bass caspase-3 sequence retains the motifs that are functionally important, such as the pentapeptide active-site motif (QACRG) and the putative cleavage sites at the aspartic acids. In the sea bass genome, the caspase-3 gene exists as a single copy gene and is organised in six exons and five introns. A very low expression of caspase-3 was detected by RT-PCR in various organs of non-stimulated sea bass, with slightly higher levels in thymus and heart and was increased in head kidneys of Photobacterium damselae ssp. piscicida infected sea bass. This increased expression was accompanied by the occurrence of high numbers of apoptoting cells with activated caspase-3.

  18. Involvement of caspases and calpains in cerebrocortical neuronal cell death is stimulus-dependent

    PubMed Central

    Moore, Jonathan D; Rothwell, Nancy J; Gibson, Rosemary M

    2002-01-01

    Caspases and calpains are mediators of apoptotic cell death. The objective of this study was to determine the role of caspases and calpains in primary cerebrocortical neuronal (CCN) death in response to a range of stimuli which reportedly induce neuronal apoptosis. Cell death of primary cultures of rat CCN was induced by staurosporine (STS), C2-ceramide (CER), camptothecin (CMT), hydrogen peroxide (H2O2) or N-methyl-D-aspartate (NMDA). Caspase and calpain activity were assessed by cleavage of α-fodrin or fluorogenic substrates. Cell death was analysed by lactate dehydrogenase (LDH) assay in the absence or presence of the pan-caspase inhibitor Boc-Asp-(OMe)-Fluoromethylketone (Baf) and/or the calpain inhibitor calpeptin (CP). Cell death induced by STS, CER or CMT was accompanied by chromatin condensation and activation of multiple caspases, particularly caspase-3-type proteases. Hydrogen peroxide (H2O2) treatment was accompanied by activation of caspases -1, -6 and -8, but not -3, whereas none of the caspases tested were activated in response to NMDA. With the exception of H2O2, when cell death was accompanied by caspase activation, it was significantly suppressed by Baf. All stimuli also induced calpain activation, but calpeptin only suppressed cell death induced by H2O2. Furthermore, co-treatment with Baf and calpeptin did not alter the cell death relative to either inhibitor alone. These findings suggest the existence of stimulus-dependent routes for the activation of caspases and calpains during death of cortical neurones and imply that although caspases and calpains are activated, their involvement in the execution of cell death varies with the stimulus. PMID:11861336

  19. Transcription Factor AP-2α Is Preferentially Cleaved by Caspase 6 and Degraded by Proteasome during Tumor Necrosis Factor Alpha-Induced Apoptosis in Breast Cancer Cells

    PubMed Central

    Nyormoi, Okot; Wang, Zhi; Doan, Dao; Ruiz, Maribelis; McConkey, David; Bar-Eli, Menashe

    2001-01-01

    Several reports have linked activating protein 2α (AP-2α) to apoptosis, leading us to hypothesize that AP-2α is a substrate for caspases. We tested this hypothesis by examining the effects of tumor necrosis factor alpha (TNF-α) on the expression of AP-2 in breast cancer cells. Here, we provide evidence that TNF-α downregulates AP-2α and AP-2γ expression posttranscriptionally during TNF-α-induced apoptosis. Both a general caspase antagonist (zVADfmk) and a caspase 6-preferred antagonist (zVEIDfmk) inhibited TNF-α-induced apoptosis and AP-2α downregulation. In vivo tests showed that AP-2α was cleaved by caspases ahead of the DNA fragmentation phase of apoptosis. Recombinant caspase 6 cleaved AP-2α preferentially, although caspases 1 and 3 also cleaved it, albeit at 50-fold or higher concentrations. Activated caspase 6 was detected in TNF-α-treated cells, thus confirming its involvement in AP-2α cleavage. All three caspases cleaved AP-2α at asp19 of the sequence asp-arg-his-asp (DRHD19). Mutating D19 to A19 abrogated AP-2α cleavage by all three caspases. TNF-α-induced cleavage of AP-2α in vivo led to AP-2α degradation and loss of DNA-binding activity, both of which were prevented by pretreatment with zVEIDfmk. AP-2α degradation but not cleavage was inhibited in vivo by PS-431 (a proteasome antagonist), suggesting that AP-2α is degraded subsequent to cleavage by caspase 6 or caspase 6-like enzymes. Cells transfected with green fluorescent protein-tagged mutant AP-2α are resistant to TNF-α-induced apoptosis, further demonstrating the link between caspase-mediated cleavage of AP-2α and apoptosis. This is the first report to demonstrate that degradation of AP-2α is a critical event in TNF-α-induced apoptosis. Since the DRHD sequence in vertebrate AP-2 is widely conserved, its cleavage by caspases may represent an important mechanism for regulating cell survival, proliferation, differentiation, and apoptosis. PMID:11438643

  20. Scopoletin induces apoptosis in human promyeloleukemic cells, accompanied by activations of nuclear factor kappaB and caspase-3.

    PubMed

    Kim, Eun-Kyung; Kwon, Kang-Beom; Shin, Byung-Cheul; Seo, Eun-A; Lee, Young-Rae; Kim, Jong-Suk; Park, Jin-Woo; Park, Byung-Hyun; Ryu, Do-Gon

    2005-07-01

    Scopoletin (6-methoxy-7-hydroxycoumarin) is a phenolic coumarin and a member of the phytoalexins. In this study we investigated whether scopoletin caused apoptosis in HL-60 promyelocytic cells and, if so, by what mechanisms. We found that scopoletin induced apoptosis as confirmed by a characteristic ladder pattern of discontinuous DNA fragments in a dose-dependent manner. The signal cascade activated by scopoletin included the heterodimeric redox-sensitive transcription factor NF-kappaB, which exhibited an upregulation of nuclear factor-kappa B (NF-kappaB) translocation to the nucleus by increase of IkappaBalpha degradation. In addition, scopoletin activated caspase-3 as was evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 resulted in the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP) to 85 kDa cleavage product in time-and dose-dependent fashions. Prior treatment of the cells with pyrrolidine dithiocarbamate, a potent inhibitor of NF-kappaB activation, or Ac-DEVD-CHO, a specific caspase-3 inhibitor, prevented scopoletin-induced caspase-3 activation, PARP cleavage, and finally DNA fragmentation. Taken together, these results suggest that scopoletin induces NF-kappaB activation, which, in turn, causes activation of caspase-3, degradation of PARP, and eventually leads to apoptotic cell death in HL-60 cells.

  1. Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells.

    PubMed

    Xiao, Fenglian; Gao, Weijie; Wang, Xiaoping; Chen, Tongsheng

    2012-06-01

    Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.

  2. Human Immunodeficiency Virus Type 1 Vpr Induces Apoptosis through Caspase Activation

    PubMed Central

    Stewart, Sheila A.; Poon, Betty; Song, Joo Y.; Chen, Irvin S. Y.

    2000-01-01

    Human immunodeficiency virus type 1 (HIV-1) Vpr is a 96-amino-acid protein that is found associated with the HIV-1 virion. Vpr induces cell cycle arrest at the G2/M phase of the cell cycle, and this arrest is followed by apoptosis. We examined the mechanism of Vpr-induced apoptosis and found that HIV-1 Vpr-induced apoptosis requires the activation of a number of cellular cysteinyl aspartate-specific proteases (caspases). We demonstrate that ectopic expression of anti-apoptotic viral proteins, which inhibit caspase activity, and addition of synthetic peptides, which represent caspase cleavage sites, can inhibit Vpr-induced apoptosis. Finally, inhibition of caspase activity and subsequent inhibition of apoptosis results in increased viral expression, suggesting that therapeutic strategies aimed at reducing Vpr-induced apoptosis in vivo require careful consideration. PMID:10708425

  3. Methyl selenium-induced vascular endothelial apoptosis is executed by caspases and principally mediated by p38 MAPK pathway.

    PubMed

    Jiang, Cheng; Kim, Ki-Hwan; Wang, Zaisen; Lü, Junxuan

    2004-01-01

    The induction of vascular endothelial cell apoptosis and inhibition of tumor-associated angiogenesis by selenium may contribute to its cancer chemopreventive effects. Here we examined the stress-activated/mitogen-activated protein kinases (p38 MAPK, ERK1/2) and protein kinase B/AKT as potential signaling mediators for apoptosis induction by a methylselenol precursor methylseleninic acid (MSeA) in human umbilical vein endothelial cells (HUVEC). Time course experiments showed that p38 MAPK hyperphosphorylation and ERK1/2 dephosphorylation occurred before the cleavage of procaspase-3 and poly(ADP-ribose) polymerase (PARP), whereas AKT dephosphorylation occurred after caspase activation. The p38 MAPK inhibitor SB202190 attenuated the MSeA-induced morphological changes and decreased DNA fragmentation and the cleavage of procaspase-3 and PARP in concordant proportions. The general caspase inhibitor zVADfmk completely blocked the MSeA-induced PARP cleavage and DNA fragmentation, whereas zDEVDfmk, an inhibitor for caspase-3-like activities, was nearly as effective for inhibiting apoptosis. In comparison, apoptosis induced by selenite in HUVECs was observed in the complete absence of an activation of the major caspases. Taken together, the data support p38 MAPK as a key upstream mediator for the methylselenol-specific induction of vascular endothelial caspase-dependent apoptosis, which is principally executed by caspase-3-like activities.

  4. Involvement of caspase-1 proteases in hypoxic brain injury. effects of their inhibitors in developing neurons.

    PubMed

    Bossenmeyer-Pourié, C; Koziel, V; Daval, J L

    2000-01-01

    To further explore the contribution of caspase-1/interleukin-1beta-convening enzyme in the consequences of hypoxia in developing brain neurons, its temporal expression profile was analysed by immunohistochemistry and western blotting in cultured neurons from the embryonic rat forebrain subjected to a hypoxic stress (95% N2/5% CO2 for 6 h), and proteolytic activity of caspase-1 was monitored as a function of time by measuring the degradation of a selective colorimetric substrate (N-acetyl-Tyr-Val-Ala-Asp-p-nitroanilide). In addition, the influence of pre- and posthypoxic treatments by caspase-1 inhibitors (N-acetyl-Tyr-Val-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone) was tested on cell outcome. Hypoxia led to delayed apoptotic neuronal death, with an elevation of the expression of both pro-caspase-1 and caspase-1 active cleavage product (ICE p20) for up to 96 h after cell reoxygenation. As reflected by cleavage of the specific substrate, caspase-1 activity progressively increased between 24 h and 96 h posthypoxia, and was blocked by inhibitors in a dose-dependent fashion. The inhibitory compounds, including when given 24 h after hypoxia, prevented neuronal death, reduced apoptosis hallmarks and also increased the number of mitotic neurons, suggesting they might promote neurogenesis. Similar observations were made when neurons were exposed to a sublethal hypoxia (i.e. 3 h). These data emphasize the participation of caspase-1 in neuronal injury consecutive to oxygen deprivation, and provide new insight into the possible cellular mechanisms by which caspase inhibitors may protect developing brain neurons.

  5. Opposing roles for caspase and calpain death proteases in L-glutamate-induced oxidative neurotoxicity

    SciTech Connect

    Elphick, Lucy M.; Hawat, Mohammad; Toms, Nick J.; Meinander, Annika; Mikhailov, Andrey; Eriksson, John E.; Kass, George E.N.

    2008-10-15

    Oxidative glutamate toxicity in HT22 murine hippocampal cells is a model for neuronal death by oxidative stress. We have investigated the role of proteases in HT22 cell oxidative glutamate toxicity. L-glutamate-induced toxicity was characterized by cell and nuclear shrinkage and chromatin condensation, yet occurred in the absence of either DNA fragmentation or mitochondrial cytochrome c release. Pretreatment with the selective caspase inhibitors either benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (pan-caspase), N-acetyl-Leu-Glu-His-Asp-aldehyde (caspase 9) or N-acetyl-Ile-Glu-Thr-Asp-aldehyde (caspase 8), significantly increased L-glutamate-induced cell death with a corresponding increase in observed nuclear shrinkage and chromatin condensation. This enhancement of glutamate toxicity correlated with an increase in L-glutamate-dependent production of reactive oxygen species (ROS) as a result of caspase inhibition. Pretreating the cells with N-acetyl-L-cysteine prevented ROS production, cell shrinkage and cell death from L-glutamate as well as that associated with the presence of the pan-caspase inhibitor. In contrast, the caspase-3/-7 inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde was without significant effect. However, pretreating the cells with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO, but not the cathepsin B inhibitor CA-074, prevented cell death. The cytotoxic role of calpains was confirmed further by: 1) cytotoxic dependency on intracellular Ca{sup 2+} increase, 2) increased cleavage of the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC and 3) immunoblot detection of the calpain-selective 145 kDa {alpha}-fodrin cleavage fragment. We conclude that oxidative L-glutamate toxicity in HT22 cells is mediated via calpain activation, whereas inhibition of caspases-8 and -9 may exacerbate L-glutamate-induced oxidative neuronal damage through increased oxidative stress.

  6. ACTIVATION OF CASPASE-3 IN THE SKELETAL MUSCLE DURING HEMODIALYSIS

    PubMed Central

    Boivin, Michel A; Battah, Shadi I; Dominic, Elizabeth A; Kalantar-Zadeh, Kamyar; Ferrando, Arny; Tzamaloukas, Antonios H; Dwivedi, Rama; Ma, Thomas A; Moseley, Pope; Raj, Dominic SC

    2010-01-01

    Background Muscle atrophy in end-stage renal disease (ESRD) may be due to the activation of apoptotic and proteolytic pathways. Objective We hypothesized that activation of caspase-3 in the skeletal muscle mediates apoptosis and proteolysis during hemodialysis (HD). Materials and Methods Eight ESRD patients were studied before (pre-HD) and during HD and the finding were compared with those from six healthy volunteers. Protein kinetics was determined by primed constant infusion of L-(ring 13C6) Phenylalanine. Results Caspase-3 activity in the skeletal muscle was higher in ESRD patients pre-HD than in controls (24966.0±4023.9 vs. 15293.3±2120.0 units, p<0.01) and increased further during HD (end-HD) (37666.6±4208.3 units) (p<0.001). 14 kDa actin fragments generated by caspase-3 mediated cleavage of actinomyosin was higher in the skeletal muscle pre-HD (68%) and during HD (164%) compared to controls. The abundance of ubiquitinized carboxy-terminal actin fragment was also significantly increased during HD. Skeletal muscle biopsies obtained at the end of HD exhibited augmented apoptosis, which was higher than that observed in pre-HD and control samples (p<0.001). IL-6 content in the soluble fraction of the muscle skeletal muscle was increased significantly during HD. Protein kinetic studies showed that catabolism was higher in ESRD patients during HD compared to pre-HD and control subjects. Muscle protein catabolism was positively associated with caspase-3 activity and skeletal muscle IL-6 content. Conclusion Muscle atrophy in ESRD may be due to IL-6 induced activation of caspase-3 resulting in apoptosis as well as muscle proteolysis during HD. PMID:20636378

  7. Lysosomal Dysfunction Promotes Cleavage and Neurotoxicity of Tau In Vivo

    PubMed Central

    Sharp, Katherine A.; Loewen, Carin A.; Mulkearns, Erin; Tyynelä, Jaana; Scherzer, Clemens R.; Feany, Mel B.

    2010-01-01

    Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer's disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer's disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer's disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer's disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D–deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer's disease. PMID:20664788

  8. Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

    PubMed Central

    Brand, Ingo L.; Civciristov, Srgjan; Taylor, Nicole L.; Talbo, Gert H.; Pantaki-Eimany, Delara; Levina, Vita; Clem, Rollie J.; Perugini, Matthew A.; Kvansakul, Marc; Hawkins, Christine J.

    2012-01-01

    Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity. PMID:22720082

  9. Mitochondria-cytochrome C-caspase-9 cascade mediates isorhamnetin-induced apoptosis.

    PubMed

    Lee, Hyo-Jung; Lee, Hyo-Jeong; Lee, Eun-Ok; Ko, Seong-Gyu; Bae, Hyun-Soo; Kim, Cheol-Ho; Ahn, Kyoo-Seok; Lu, Junxuan; Kim, Sung-Hoon

    2008-11-08

    Isorhamnetin is a flavanoid present in plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. Since the plasma level of isorhamnetin is maintained longer than quercetin, isorhamnetin may be a key metabolite to mediate the anti-tumor effect of quercetin. In the present study, we investigated the apoptotic mechanism of isorhamnetin in Lewis lung cancer (LLC) cells in vitro and established its in vivo anti-cancer efficacy. In cell culture, isorhamnetin significantly increased DNA fragmentation, and TUNEL positive apoptotic bodies and sub-G(1) apoptotic population in time- and dose-dependent manners. Western blot analyses revealed increased cleavage of caspase-3, and caspase-9 and PARP and increased cytosolic cytochrome C in isorhamnetin-treated cells. These events were accompanied by a reduced mitochondrial potential. Apoptosis was blocked by a general caspase inhibitor or the specific inhibitor of caspase-3 or -9. These in vitro results support mitochondria-dependent caspase activation to mediate isorhamnetin-induced apoptosis. Furthermore, an animal study revealed for the first time that isorhamnetin given by i.p. injection at a dose that is at least one order of magnitude lower than quercetin significantly suppressed the weights of tumors excised from LLC bearing mice. The in vivo anti-tumor efficacy was accompanied by increased TUNEL-positive and cleaved-caspase-3-positive tumor cells. Our data therefore support isorhamnetin as an active anti-cancer metabolite of quercetin in part through caspase-mediated apoptosis.

  10. The IAP-antagonist ARTS initiates caspase activation upstream of cytochrome C and SMAC/Diablo

    PubMed Central

    Edison, N; Zuri, D; Maniv, I; Bornstein, B; Lev, T; Gottfried, Y; Kemeny, S; Garcia-Fernandez, M; Kagan, J; Larisch, S

    2012-01-01

    ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis. PMID:21869827

  11. Raf-1 Activation Prevents Caspase 9 Processing Downstream of Apoptosome Formation

    PubMed Central

    Cagnol, Sébastien; Mansour, Anna; Van Obberghen-Schilling, Ellen; Chambard, Jean-Claude

    2011-01-01

    In many cell types, growth factor removal induces the release of cytochrome-c from mitochondria that leads to activation of caspase-9 in the apoptosome complex. Here, we show that sustained stimulation of the Raf-1/MAPK1,3 pathway prevents caspase-9 activation induced by serum depletion in CCL39/ΔRaf-1:ER fibroblasts. The protective effect mediated by Raf-1 is sensitive to MEK inhibition that is sufficient to induce caspase-9 cleavage in exponentially growing cells. Raf-1 activation does not inhibit the release of cytochrome-c from mitochondria while preventing caspase-9 activation. Gel filtration chromatography analysis of apoptosome formation in cells shows that Raf-1/MAPK1,3 activation does not interfere with APAF-1 oligomerization and recruitment of caspase 9. Raf-1-mediated caspase-9 inhibition is sensitive to emetine, indicating that the protective mechanism requires protein synthesis. However, the Raf/MAPK1,3 pathway does not regulate XIAP. Taken together, these results indicate that the Raf-1/MAPK1,3 pathway controls an apoptosis regulator that prevents caspase-9 activation in the apoptosome complex. PMID:21637382

  12. Axonal Cleaved Caspase-3 Regulates Axon Targeting and Morphogenesis in the Developing Auditory Brainstem

    PubMed Central

    Rotschafer, Sarah E.; Allen-Sharpley, Michelle R.; Cramer, Karina S.

    2016-01-01

    Caspase-3 is a cysteine protease that is most commonly associated with cell death. Recent studies have shown additional roles in mediating cell differentiation, cell proliferation and development of cell morphology. We investigated the role of caspase-3 in the development of chick auditory brainstem nuclei during embryogenesis. Immunofluorescence from embryonic days E6–13 revealed that the temporal expression of cleaved caspase-3 follows the ascending anatomical pathway. The expression is first seen in the auditory portion of VIIIth nerve including central axonal regions projecting to nucleus magnocellularis (NM), then later in NM axons projecting to nucleus laminaris (NL), and subsequently in NL dendrites. To examine the function of cleaved caspase-3 in chick auditory brainstem development, we blocked caspase-3 cleavage in developing chick embryos with the caspase-3 inhibitor Z-DEVD-FMK from E6 to E9, then examined NM and NL morphology and NM axonal targeting on E10. NL lamination in treated embryos was disorganized and the neuropil around NL contained a significant number of glial cells normally excluded from this region. Additionally, NM axons projected into inappropriate portions of NL in Z-DEVD-FMK treated embyros. We found that the presence of misrouted axons was associated with more severe NL disorganization. The effects of axonal caspase-3 inhibition on both NL morphogenesis and NM axon targeting suggest that these developmental processes are coordinated, likely through communication between axons and their targets. PMID:27822180

  13. Kaurene diterpene induces apoptosis in human leukemia cells partly through a caspase-8-dependent pathway.

    PubMed

    Kondoh, Masuo; Suzuki, Ikue; Sato, Masao; Nagashima, Fumihiro; Simizu, Siro; Harada, Motoki; Fujii, Makiko; Osada, Hiroyuki; Asakawa, Yoshinori; Watanabe, Yoshiteru

    2004-10-01

    Defects in apoptosis signaling pathways contribute to tumorigenesis and drug resistance, and these defects are often a cause of failure of chemotherapy. Thus, a major goal in chemotherapy is to find cytotoxic agents that restore the ability of tumor cells to undergo apoptosis. We previously found that an Ent-kaurene diterpene, Ent-11alpha-hydroxy-16-kauren-15-one (KD), induced apoptosis in human promyelocytic leukemia HL-60 cells. Here, we found that caspase-8, an apoptotic factor, is involved in KD-induced apoptosis. Although treatment of HL-60 cells with KD resulted in the activation of caspase-8 and -9, a caspase-8-specific inhibitor but not a caspase-9-specific inhibitor attenuated KD-induced apoptosis. Expression of a catalytically inactive caspase-8 partly attenuated KD-induced apoptosis. Treatment with KD led to a time-dependent cleavage of Bid, a substrate of caspase-8, as well as to the proteolytic processing of procaspase-8, indicating that KD treatment induces apoptosis through a caspase-8-dependent pathway. Moreover, overexpression of the drug resistance factor Bcl-2, which is frequently overexpressed in many tumors, failed to confer resistance to KD-induced cytotoxicity. Thus, KD may be a promising experimental cytotoxic agent that possibly points to new strategies to overcome a drug resistance.

  14. Mollugin induces apoptosis in human Jurkat T cells through endoplasmic reticulum stress-mediated activation of JNK and caspase-12 and subsequent activation of mitochondria-dependent caspase cascade regulated by Bcl-xL

    SciTech Connect

    Kim, Sun Mi; Park, Hae Sun; Jun, Do Youn; Woo, Hyun Ju; Woo, Mi Hee; Yang, Chae Ha; Kim, Young Ho

    2009-12-01

    Exposure of Jurkat T cells to mollugin (15-30 muM), purified from the roots of Rubia cordifolia L., caused cytotoxicity and apoptotic DNA fragmentation along with mitochondrial membrane potential disruption, mitochondrial cytochrome c release, phosphorylation of c-Jun N-terminal kinase (JNK), activation of caspase-12, -9, -7, -3, and -8, cleavage of FLIP and Bid, and PARP degradation, without accompanying necrosis. While these mollugin-induced cytotoxicity and apoptotic events including activation of caspase-8 and mitochondria-dependent activation of caspase cascade were completely prevented by overexpression of Bcl-xL, the activation of JNK and caspase-12 was prevented to much lesser extent. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk), the caspase-9 inhibitor (z-LEHD-fmk), the caspase-3 inhibitor (z-DEVD-fmk) or the caspase-12 inhibitor (z-ATAD-fmk) at the minimal concentration to prevent mollugin-induced apoptosis appeared to completely block the activation of caspase-7 and -8, and PARP degradation, but failed to block the activation of caspase-9 and -3 with allowing a slight enhancement in the level of JNK phosphorylation. Both FADD-positive wild-type Jurkat clone A3 and FADD-deficient Jurkat clone I2.1 exhibited a similar susceptibility to the cytotoxicity of mollugin, excluding involvement of Fas/FasL system in triggering mollugin-induced apoptosis. Normal peripheral T cells were more refractory to the cytotoxicity of mollugin than were Jurkat T cells. These results demonstrated that mollugin-induced cytotoxicity in Jurkat T cells was mainly attributable to apoptosis provoked via endoplasmic reticulum (ER) stress-mediated activation of JNK and caspase-12, and subsequent mitochondria-dependent activation of caspase-9 and -3, leading to activation of caspase-7 and -8, which could be regulated by Bcl-xL.

  15. Old, new and emerging functions of caspases

    PubMed Central

    Shalini, S; Dorstyn, L; Dawar, S; Kumar, S

    2015-01-01

    Caspases are proteases with a well-defined role in apoptosis. However, increasing evidence indicates multiple functions of caspases outside apoptosis. Caspase-1 and caspase-11 have roles in inflammation and mediating inflammatory cell death by pyroptosis. Similarly, caspase-8 has dual role in cell death, mediating both receptor-mediated apoptosis and in its absence, necroptosis. Caspase-8 also functions in maintenance and homeostasis of the adult T-cell population. Caspase-3 has important roles in tissue differentiation, regeneration and neural development in ways that are distinct and do not involve any apoptotic activity. Several other caspases have demonstrated anti-tumor roles. Notable among them are caspase-2, -8 and -14. However, increased caspase-2 and -8 expression in certain types of tumor has also been linked to promoting tumorigenesis. Increased levels of caspase-3 in tumor cells causes apoptosis and secretion of paracrine factors that promotes compensatory proliferation in surrounding normal tissues, tumor cell repopulation and presents a barrier for effective therapeutic strategies. Besides this caspase-2 has emerged as a unique caspase with potential roles in maintaining genomic stability, metabolism, autophagy and aging. The present review focuses on some of these less studied and emerging functions of mammalian caspases. PMID:25526085

  16. Caspase-3 is required in the apoptotic disintegration of the nuclear matrix

    SciTech Connect

    Kivinen, Katri; Kallajoki, Markku; Taimen, Pekka . E-mail: pekka.taimen@utu.fi

    2005-11-15

    Apoptotic breakdown of cellular structures is largely mediated by caspases. One target of degradation is a proteinaceous framework of the nucleus termed the nuclear matrix. We compared the apoptotic changes of the nuclear matrix in staurosporine-treated caspase-3-deficient MCF-7 cells transfected with intact CASP-3 gene (MCF-7c3) or an empty vector (MCF-7v) as a control. Nuclear Mitotic Apparatus protein (NuMA), lamin A/C and lamin B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. In both cell lines, staurosporine induced rapid cytoplasmic shrinkage and partial chromatin condensation. MCF-7c3 cells formed apoptotic bodies, whereas MCF-7v cells did not. NuMA and lamins were actively cleaved in MCF-7c3 cells following caspase-3 activation, but only minimal or no cleavage was detected in MCF-7v cells. Interestingly, lamin B but not lamin A/C was relocated into cytoplasmic granules in apoptotic MCF-7v cells. Pancaspase inhibitor, z-VAD-fmk, prevented the apoptotic changes, while caspase-3 inhibitor, z-DEVD-fmk, induced lamin B granules in both cell lines. These results show that caspase-3 is involved in the cleavage of NuMA and lamins either directly or by activating other proteases. This may be essential for disintegration of the nuclear structure during apoptosis.

  17. Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy.

    PubMed

    Nandi, Nilay; Tyra, Lauren K; Stenesen, Drew; Krämer, Helmut

    2014-10-27

    How cellular stresses up-regulate autophagy is not fully understood. One potential regulator is the Drosophila melanogaster protein Acinus (Acn), which is necessary for autophagy induction and triggers excess autophagy when overexpressed. We show that cell type-specific regulation of Acn depends on proteolysis by the caspase Dcp-1. Basal Dcp-1 activity in developing photoreceptors is sufficient for this cleavage without a need for apoptosis to elevate caspase activity. On the other hand, Acn was stabilized by loss of Dcp-1 function or by the presence of a mutation in Acn that eliminates its conserved caspase cleavage site. Acn stability also was regulated by AKT1-mediated phosphorylation. Flies that expressed stabilized forms of Acn, either the phosphomimetic Acn(S641,731D) or the caspase-resistant Acn(D527A), exhibited enhanced basal autophagy. Physiologically, these flies showed improvements in processes known to be autophagy dependent, including increased starvation resistance, reduced Huntingtin-induced neurodegeneration, and prolonged life span. These data indicate that AKT1 and caspase-dependent regulation of Acn stability adjusts basal autophagy levels.

  18. Cross-talk between the calpain and caspase-3 proteolytic systems in the diaphragm during prolonged mechanical ventilation

    PubMed Central

    Nelson, W. Bradley; Ashley J., Smuder; Hudson, Matthew B.; Talbert, Erin E.; Powers, Scott K.

    2012-01-01

    OBJECTIVE Diaphragmatic weakness, due to both atrophy and contractile dysfunction, is a well-documented response following prolonged mechanical ventilation (MV). Evidence indicates that activation of the proteases calpain and caspase-3 are essential for MV-induced diaphragmatic weakness to occur. We tested the hypothesis that a regulatory cross-talk exists between calpain and caspase-3 in the diaphragm during prolonged MV. To test this prediction, we determined if selective pharmacological inhibition of calpain would prevent activation of caspase-3 and conversely, if selective inhibition of caspase-3 would abate calpain activation. DESIGN Animal study. SETTING University Research Laboratory SUBJECTS Female Sprague-Dawley rats INTERVENTIONS Animals were randomly divided into a control or one of three 12 hour MV groups that were treated with/without a selective pharmacological protease inhibitor: 1) control; 2) MV; 3) MV with a selective caspase-3 inhibitor; and 4) MV with a selective calpain inhibitor. MEASUREMENTS AND MAIN RESULTS Compared to control, MV resulted in calpain and caspase-3 activation in the diaphragm accompanied by atrophy of type I, type IIa, and type IIx/IIb fibers. Independent inhibition of either calpain or caspase-3 prevented this MV-induced atrophy. Pharmacological inhibition of calpain prevented MV-induced activation of diaphragmatic caspase-3 and inhibition of caspase-3 prevented activation of diaphragmatic calpain. Further, calpain inhibition also prevented the activation of caspase-9 and caspase-12, along with the cleavage of Bid to tBid, all upstream signals for caspase-3 activation. Lastly, caspase-3 inhibition prevented the MV-induced degradation of the endogenous calpain inhibitor, calpastatin. CONCLUSIONS Collectively, these results indicate that MV-induced diaphragmatic atrophy is dependent upon the activation of both calpain and caspase-3. Importantly, these findings provide the first experimental evidence in diaphragm muscle that

  19. Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

    PubMed Central

    Oropesa-Ávila, M; Fernández-Vega, A; de la Mata, M; Maraver, J G; Cordero, M D; Cotán, D; de Miguel, M; Calero, C P; Paz, M V; Pavón, A D; Sánchez, M A; Zaderenko, A P; Ybot-González, P; Sánchez-Alcázar, J A

    2013-01-01

    Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na+/Ca2+ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na+/K+ pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential

  20. HIV-1 viral protein r induces ERK and caspase-8 dependent apoptosis in renal tubular epithelial cells

    PubMed Central

    Snyder, Alexandra; Alsauskas, Zygimantas C.; Leventhal, Jeremy S.; Rosenstiel, Paul E.; Gong, Pengfei; Chan, Justin JK; Barley, Kevin; He, John C.; Klotman, Mary E.; Ross, Michael J.; Klotman, Paul E.

    2010-01-01

    Objective HIV-associated nephropathy is the most common cause of end stage renal disease in persons with HIV/AIDS and is characterized by focal glomerulosclerosis and dysregulated renal tubular epithelial cell (RTEC) proliferation and apoptosis. HIV-1 viral protein r (Vpr) has been implicated in HIV-induced RTEC apoptosis but the mechanisms of Vpr-induced RTEC apoptosis are unknown. The aim of this study was therefore to determine the mechanisms of Vpr-induced apoptosis in RTEC. Methods Apoptosis and caspase activation were analyzed in human RTEC cells (HK2) after transduction with Vpr-expressing and control lentiviral vectors. Bax and BID were inhibited with lentiviral shRNA, and ERK activation was blocked with the MEK1,2 inhibitor, U0126. Results Vpr induced apoptosis as indicated by caspase 3/7 activation, PARP-1 cleavage and mitochondrial injury. Vpr activated both caspases-8 and 9. Inhibition of Bax reduced Vpr-induced apoptosis, as reported in other cell types. Additionally, Vpr induced cleavage of BID to tBID and suppression of BID expression prevented Vpr-induced apoptosis. Since sustained ERK activation can activate caspase-8 in some cell types, we studied the role of ERK in Vpr-induced caspase-8 activation. Vpr induced sustained ERK activation in HK2 cells and incubation with U0126 reduced Vpr-induced caspase-8 activation, BID cleavage and apoptosis. We detected phosphorylated ERK in RTEC in HIVAN biopsy specimens by immunohistochemistry. Conclusions These studies delineate a novel pathway of Vpr-induced apoptosis in RTEC, which is mediated by sustained ERK activation, resulting in caspase 8-mediated cleavage of BID to tBID, thereby facilitating Bax-mediated mitochondrial injury and apoptosis. PMID:20404718

  1. Intracellular water motion decreases in apoptotic macrophages after caspase activation.

    PubMed

    Hortelano, S; García-Martín, M L; Cerdán, S; Castrillo, A; Alvarez, A M; Boscá, L

    2001-10-01

    Triggering of the macrophage cell line RAW 264.7 with lipopolysaccharide and interferon-gamma promoted apoptosis that was prevented by inhibitors of type 2 nitric oxide synthase or caspase. Using (1)H NMR analysis, we have investigated the changes of the intracellular transverse relaxation time (T(2)) and apparent diffusion coefficient (ADC) as parameters reflecting the rotational and translational motions of water in apoptotic macrophages. T(2) values decreased significantly from 287 to 182 ms in cells treated for 18 h with NO-donors. These changes of T(2) were prevented by caspase inhibitors and were not due to mitochondrial depolarization or microtubule depolymerization. The decrease of the intracellular values of T(2) and ADC in apoptotic macrophages was observed after caspase activation, but preceded phosphatidylserine exposure and nucleosomal DNA cleavage. The changes of water motion were accompanied by an enhancement of the hydrophobic properties of the intracellular milieu, as detected by fluorescent probes. These results indicate the occurrence of an alteration in the physicochemical properties of intracellular water during the course of apoptosis.

  2. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35

    PubMed Central

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-01-01

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its ‘reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro. PMID:22170098

  3. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

    PubMed

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-12-15

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro.

  4. Caspase-10 Is the Key Initiator Caspase Involved in Tributyltin-Mediated Apoptosis in Human Immune Cells

    PubMed Central

    Krug, Harald F.

    2012-01-01

    Tributyltin (TBT) is one of the most toxic compounds produced by man and distributed in the environment. A multitude of toxic activities have been described, for example, immunotoxic, neurotoxic, and endocrine disruptive effects. Moreover, it has been shown for many cell types that they undergo apoptosis after treatment with TBT and the cell death of immune cells could be the molecular background of its immunotoxic effect. As low as 200 nM up to 1 μM of TBT induces all signs of apoptosis in Jurkat T cells within 1 to 24 hrs of treatment. When compared to Fas-ligand control stimulation, the same sequence of events occurs: membrane blebbing, phosphatidylserine externalisation, the activation of the “death-inducing signalling complex,” and the following sequence of cleavage processes. In genetically modified caspase-8-deficient Jurkat cells, the apoptotic effects are only slightly reduced, whereas, in FADD-negative Jurkat cells, the TBT effect is significantly diminished. We could show that caspase-10 is recruited by the TRAIL-R2 receptor and apoptosis is totally prevented when caspase-10 is specifically inhibited in all three cell lines. PMID:22287961

  5. Caspase-resistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria.

    PubMed

    Nguyen, M; Breckenridge, D G; Ducret, A; Shore, G C

    2000-09-01

    BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two identical caspase recognition sites (AAVD.G) that are preferentially cleaved by initiator caspases, including caspase 8. Cleavage of BAP31 during apoptosis generates a p20 fragment that remains integrated in the membrane and, when expressed ectopically, is a potent inducer of cell death. To examine the consequences of maintaining the structural integrity of BAP31 during apoptosis, the caspase recognition aspartate residues were mutated to alanine residues, and Fas-mediated activation of caspase 8 and cell death were examined in human KB epithelial cells stably expressing the caspase-resistant mutant crBAP31. crBAP31 only modestly slowed the time course for activation of caspases, as assayed by the processing of procaspases 8 and 3 and the measurement of total DEVDase activity. As a result, cleavage of the caspase targets poly(ADP-ribosyl) polymerase and endogenous BAP31, as well as the redistribution of phosphatidylserine and fragmentation of DNA, was observed. In contrast, cytoplasmic membrane blebbing and fragmentation and apoptotic redistribution of actin were strongly inhibited, cell morphology was retained near normal, and the irreversible loss of cell growth potential following removal of the Fas stimulus was delayed. Of note, crBAP31-expressing cells also resisted Fas-mediated release of cytochrome c from mitochondria, and the mitochondrial electrochemical potential was only partly reduced. These results argue that BAP31 cleavage is important for manifesting cytoplasmic apoptotic events associated with membrane fragmentation and reveal an unexpected cross talk between mitochondria and the endoplasmic reticulum during Fas-mediated apoptosis in vivo.

  6. Acid-dependent Interleukin-1 (IL-1) Cleavage Limits Available Pro-IL-1β for Caspase-1 Cleavage.

    PubMed

    Edye, Michelle E; Brough, David; Allan, Stuart M

    2015-10-16

    Noncommunicable diseases such as cardiovascular disease (stroke and heart attack), cancer, chronic respiratory disease, and diabetes are a leading cause of death and disability worldwide and are worsened by inflammation. IL-1 is a driver of inflammation and implicated in many noncommunicable diseases. Acidosis is also a key feature of the inflammatory microenvironment; therefore it is vital to explore IL-1 signaling under acidic conditions. A HEK-IL-1 reporter assay and brain endothelial cell line were used to explore activity of mature IL-1α and IL-1β at pH 7.4 and pH 6.2, an acidic pH that can be reached under inflammatory or ischemic conditions, alongside cathepsin D-cleaved 20-kDa IL-1β produced under acidic conditions. We report that mature IL-1 signaling at IL-1 receptor type 1 (IL-1R1) is maintained at pH 6.2, but the activity of the decoy receptor, IL-1R2, is reduced. Additionally, cathepsin D-cleaved 20-kDa IL-1β was minimally active at IL-1R1 and was not further cleaved to highly active 17-kDa IL-1β. Therefore formation of the 20-kDa form of IL-1β may prevent the generation of mature bioactive IL-1β and thus may limit inflammation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death

    PubMed Central

    DeLaney, Alexandra; Santos-Marrero, Melanie; Grier, Jennifer T.; Sun, Yan; Zwack, Erin E.; Hu, Baofeng; Olsen, Tayla M.; Rongvaux, Anthony; López, Carolina B.; Oberst, Andrew; Beiting, Daniel P.; Brodsky, Igor E.

    2016-01-01

    Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense. PMID:27737018

  8. Development of a bidirectional caspase-3 expression system for the induction of apoptosis.

    PubMed

    White, Martyn K; Amini, Shohreh; Khalili, Kamel; Kogan, Michael; Donaldson, Keri; Darbinian, Nune

    2008-06-01

    Caspase-3 is the executioner caspase of apoptosis whose activation in mammalian cells represents the last stage of the programmed cell death signaling pathway and the initiation of the lethal digestion of cell proteins. Active caspase-3 is a tetramer composed of two p12 and two p17 subunits derived from cleavage of procaspase-3 during activation. Here, we armed GFP-fusion proteins of both the caspase-3 p12 and p17 subunits with signals from Ig-kappa light chain that allows its efficient secretion from the cells (Sec) and from HIV-1 Tat that facilitates its uptake and nuclear translocation by other cells (NLS). We found that treatment of cells with conditioned media from cells expressing both Sec-GFP-p17-NLS and Sec-GFP-p12-NLS was able to transduce active caspase-3 with consequent cell death of treated cultures. Use of various combinations of constructs demonstrated that both subunits were required and that each one needed to possess both Sec and NLS. Our observations introduce a bidirectional protein transduction system with the ability to introduce active caspase-3 into cells and cause apoptosis. This system may have important therapeutic applications.

  9. Rapid induction of mitochondrial events and caspase-independent apoptosis in Survivin-targeted melanoma cells

    PubMed Central

    Liu, Tong; Brouha, Brook; Grossman, Douglas

    2008-01-01

    The inhibitor of apoptosis (IAP) protein Survivin is expressed in most cancers and is a key factor in maintaining apoptosis resistance. Although several IAPs have been shown to act as direct inhibitors of caspases, the precise antiapoptotic function of Survivin remains controversial. To clarify the mechanism by which Survivin protects cells, we investigated the kinetics of apoptosis and apoptotic events following Survivin inhibition utilizing a melanoma cell line harboring a tetracycline-regulated Survivin dominant-negative mutant (Survivin-T34A). Blocking Survivin resulted in both caspase activation and apoptosis; however, the level of apoptosis was only partially reduced by caspase inhibition. Survivin blockade also resulted in mitochondrial events that preceded caspase activation, including depolarization and release of cytochrome c and Smac/DIABLO. Levels of other IAPs were not altered in Survivin-targeted cells, although modest cleavage of XIAP and Livin was observed. The earliest proapoptotic event observed in Survivin-targeted cells was nuclear translocation of mitochondrial apoptosis-inducing factor (AIF), known to trigger both apoptotic mitochondrial events and caspase-independent DNA fragmentation. These findings suggest that a key anti-apoptotic function of Survivin relates to inhibition of mitochondrial and AIF-dependent apoptotic pathways, and its expression in melanoma and other cancers likely protects against both caspase-independent and -dependent apoptosis. PMID:14712209

  10. Phytosphingosine induces apoptotic cell death via caspase 8 activation and Bax translocation in human cancer cells.

    PubMed

    Park, Moon-Taek; Kang, Jung A; Choi, Jung-A; Kang, Chang-Mo; Kim, Tae-Hwan; Bae, Sangwoo; Kang, Seongman; Kim, Sujong; Choi, Weon-Ik; Cho, Chul-Koo; Chung, Hee-Yong; Lee, Yun-Sil; Lee, Su-Jae

    2003-02-01

    Sphingolipid metabolites, such as sphingosine and ceramide, are highly bioactive compounds and are involved in diverse cell processes, including cell-cell interaction, cell proliferation, differentiation, and apoptosis. However, the physiological roles of phytosphingosine are poorly understood. In this study, we report that phytosphingosine can potently induce apoptotic cell death in human cancer cells via caspase activation and caspase-independent cytochrome c release. Phytosphingosine-induced apoptosis was determined by Hoechst 33258 staining, flow cytometric analysis, and DNA fragmentation assay. Involvement of caspases was determined by immunoblot analysis and cell death detection assays after treatment with synthetic inhibitor z-Val-Ala-Asp-fluoromethyl ketone, z-DEVD-fmk, or z-IETD-fmk. Death receptor (DR) dependency was analyzed by examining expression of DRs (Fas, DR4, DR5, TNFR1, and R2), and interaction of Fas-associated death domain and caspase 8. Involvement of the mitochondria pathway was examined by monitoring of the mitochondria membrane potential, cytochrome c release, and Bax translocation. Phytosphingosine-treated cells displayed several features of apoptosis, including increase of sub-G(1) population, DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. We observed that phytosphingosine cause activation of caspase 8 in a DR-independent fashion. Phytosphingosine also induced activation of caspase 9 and 3, loss of mitochondrial membrane potential, and the cytochrome c release from mitochondria. However, we failed to detect Bid cleavage. Moreover, caspase 8 inhibitor z-IETD-fmk did not affect phytosphingosine-induced cytochrome c release and caspase 9 activation, suggesting that phytosphingosine-induced cytochrome c release is caused by caspase 8-independent manner. Phytosphingosine induced mitochondrial translocation of Bax from the cytosol without changes in the protein levels of Bcl-2, Bcl-xL, and Bax. In addition, Bcl-2/Bax interaction

  11. Induction of caspase-dependent and -independent apoptosis in response to methionine restriction.

    PubMed

    Lu, Shan; Hoestje, Sara M; Choo, Eugene; Epner, Daniel E

    2003-02-01

    Tumor cells are more sensitive to methionine restriction than normal tissues, a phenomenon known as methionine auxotrophy. Previous studies have demonstrated that methionine restriction causes tumor cell growth arrest and eventually apoptosis. The current studies were undertaken to elucidate the molecular pathways leading to apoptosis induced by methionine restriction. We found that methionine restriction induced formation of oligonucleosomal DNA fragment and cytochrome c release from mitochondria in methionine-dependent PC3 and Hela cells. Methionine restriction also led to cleavage and activation of initiator and effector caspases in Hela cells but not PC3 cells. Furthermore, methionine restriction resulted in cleavage of BID and reduction in Bcl-2 levels in both cell lines. These data suggest that apoptosis induced by methionine restriction is mitochondria-dependent. Methionine restriction induced caspase-independent cell death in PC3 cells, whereas it stimulated caspase-dependent cell death in Hela cells. Cleavage of BID and decreased expression of Bcl-2 upon methionine deprivation may be the underlying mechanism to stimulate release of cytochrome c from mitochondria.

  12. A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis

    SciTech Connect

    Walters, Jad; Pop, Cristina; Scott, Fiona L.; Drag, Marcin; Swartz, Paul; Mattos, Carla; Salvesen, Guy S.; Clark, A.Clay

    2010-03-12

    The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis). The 1.63 {angstrom} (1 {angstrom} = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3. Structural modelling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to form in the procaspase in the absence of cleavage. The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.

  13. Imaging of activated caspase-3 in living cell by fluorescence resonance energy transfer during photosensitization-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Wu, Yunxia; Xing, Da; Chen, Qun; Tang, Yonghong

    2005-01-01

    Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.

  14. Caspase 3 promotes genetic instability and carcinogenesis

    PubMed Central

    Liu, Xinjian; He, Yujun; Li, Fang; Huang, Qian; Kato, Takamitsu A.; Hall, Russell P; Li, Chuan-Yuan

    2015-01-01

    Summary Apoptosis is typically considered an anti-oncogenic process since caspase activation can promote the elimination of genetically unstable or damaged cells. We report that a central effector of apoptosis, caspase 3, facilitates, rather than suppresses, chemical and radiation-induced genetic instability and carcinogenesis. We found that a significant fraction of mammalian cells treated with ionizing radiation can survive, despite caspase 3 activation. Moreover, this sublethal activation of caspase 3 promoted persistent DNA damage and oncogenic transformation. In addition, chemically-induced skin carcinogenesis was significantly reduced in mice genetically deficient in caspase 3. Furthermore, attenuation of Endo G activity significantly reduced radiation-induced DNA damage and oncogenic transformation, identifying Endo G as a downstream effector of caspase 3 in this pathway. Our findings suggest that rather than acting as a broad inhibitor of carcinogenesis, caspase 3 activation may contribute to genome instability and play a pivotal role in tumor formation following damage. PMID:25866249

  15. Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly

    PubMed Central

    Graumann, Kristin; Schaumburg, Frieder; Reubold, Thomas F.; Hippe, Diana; Eschenburg, Susanne; Lüder, Carsten G. K.

    2015-01-01

    Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome c in vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway. PMID:28357287

  16. Selenite-induced p53 Ser-15 phosphorylation and caspase-mediated apoptosis in LNCaP human prostate cancer cells.

    PubMed

    Jiang, Cheng; Hu, Hongbo; Malewicz, Barbara; Wang, Zaisen; Lü, Junxuan

    2004-07-01

    The issue of p53 requirement for the caspase-mediated apoptosis induced by selenium in a cancer chemoprevention or chemotherapy context has not been critically addressed. We and others have shown that selenite induces apoptotic DNA laddering in the p53-mutant DU145 prostate cancer cells and the p53-null HL60 leukemia cells without the cleavage of poly(ADP-ribose) polymerase (PARP; i.e., caspase-independent apoptosis), whereas selenium compounds leading to the formation of methylselenol induce caspase-mediated apoptosis in these cells. Because selenite induces DNA single strand breaks, and because certain types of DNA damage activate p53, we investigated whether the human LNCaP prostate cancer cells, which contain a wild-type p53, execute selenite-induced apoptosis through caspase pathways. The results showed that exposure of LNCaP cells for 24 hours to lower micromolar concentrations of selenite led to DNA laddering, and to the cleavage of PARP and several pro-caspases. In contrast to this apoptosis sensitivity, LNCaP cells were rather resistant to similar concentrations of the methylselenol precursor methylseleninic acid. Selenite treatment led to a significant increase in p53 phosphorylation on Ser-15 (Ser15P). Time course experiments showed that p53 Ser15P occurred several hours before caspase activation and PARP cleavage. The general caspase inhibitor zVADfmk completely blocked PARP cleavage, and significantly decreased DNA laddering, but did not affect p53 Ser15P. An inhibitor for caspase-8 was equally as protective as that for caspase-9 against the selenite-induced apoptosis. Attenuating p53 by a chemical inhibitor pifithrin-alpha decreased the selenite-induced p53 Ser15P and led to concordant reductions of PARP cleavage and apoptosis. In summary, selenite-induced p53 Ser15P appeared to be important for activating the caspase-mediated apoptosis involving both the caspase-8 and the caspase-9 pathways in the LNCaP cells.

  17. TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

    SciTech Connect

    Polek, Tara C.; Talpaz, Moshe; Spivak-Kroizman, Taly R. . E-mail: tspivakk@mdanderson.org

    2006-10-27

    Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects.

  18. Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.

    PubMed Central

    Stennicke, H R; Renatus, M; Meldal, M; Salvesen, G S

    2000-01-01

    Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences. PMID:10947972

  19. Saturated fatty acids activate caspase-4/5 in human monocytes, triggering IL-1β and IL-18 release.

    PubMed

    Pillon, Nicolas J; Chan, Kenny L; Zhang, Shitian; Mejdani, Marios; Jacobson, Maya R; Ducos, Alexandre; Bilan, Philip J; Niu, Wenyan; Klip, Amira

    2016-11-01

    Obesity is associated with metabolic tissue infiltration by monocyte-derived macrophages. Saturated fatty acids contribute to proinflammatory gene induction in tissue-embedded immune cells. However, it is unknown how circulating monocytes, the macrophage precursors, react to high-fat environments. In macrophages, saturated fatty acids activate inflammatory pathways and, notably, prime caspase-associated inflammasomes. Inflammasome-activated IL-1β contributes to type 2 diabetes. We hypothesized that 1) human monocytes from obese patients show caspase activation, and 2) fatty acids trigger this response and consequent release of IL-1β/IL-18. Human peripheral blood monocytes were sorted by flow cytometry, and caspase activity was measured with a FLICA dye-based assay. Blood monocytes from obese individuals exhibited elevated caspase activity. To explore the nature and consequence of this activity, human THP1 monocytes were exposed to saturated or unsaturated fatty acids. Caspase activity was revealed by isoform-specific cleavage and enzymatic activity; cytokine expression/release was measured by qPCR and ELISA. Palmitate, but not palmitoleate, increased caspase activity in parallel to the release of IL-1β and IL-18. Palmitate induced eventual monocyte cell death with features of pyroptosis (an inflammation-linked cell death program involving caspase-4/5), scored through LDH release, vital dye influx, cell volume changes, and nuclear morphology. Notably, selective gene silencing or inhibition of caspase-4/5 reduced palmitate-induced release of IL-1β and IL-18. In summary, monocytes from obese individuals present elevated caspase activity. Mechanistically, palmitate activates a pyroptotic program in monocytes through caspase-4/5, causing inflammatory cytokine release, additional to inflammasomes. These caspases represent potential, novel, therapeutic targets to taper obesity-associated inflammation. Copyright © 2016 the American Physiological Society.

  20. Phylogenomics of caspase-activated DNA fragmentation factor

    SciTech Connect

    Eckhart, Leopold . E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz; Tschachler, Erwin

    2007-04-27

    The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans.

  1. The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis.

    PubMed Central

    Mahoney, James A; Odin, Joseph A; White, Sarah M; Shaffer, David; Koff, Andrew; Casciola-Rosen, Livia; Rosen, Antony

    2002-01-01

    In the present study, we demonstrate that a human homologue of Ufd2p (a yeast protein that catalyses the formation of long polyubiquitin chains, and is implicated in responses to environmental stress), UFD2 (ubiquitin fusion degradation protein-2), is cleaved during apoptosis induced by multiple stimuli, including UVB irradiation, Fas ligation, staurosporine treatment and cytotoxic lymphocyte granule-induced death. Caspase 6 and granzyme B efficiently cleave UFD2 [k(cat)/K(m)=(4-5) x 10(4) M(-1) x s(-1)] at Asp(123), whereas caspases 3 and 7 cleave UFD2 approx. 10-fold less efficiently immediately upstream at Asp(109). Thus UFD2 is added to the growing list of proteins with closely spaced caspase and granzyme B cleavage sites, suggesting the presence of a previously unrecognized, conserved motif. Both cleavage sites are contained and conserved within a novel 300-amino-acid N-terminal domain present in apparent UFD2 orthologues in mice and zebrafish, but absent in all UFD2 family members in lower eukaryotes. Full-length recombinant UFD2 exhibited ubiquitin-protein ligase ('E3')-like ubiquitination activity in vitro, but this activity was abolished in recombinant UFD2 truncated at the granzyme B/caspase 6 cleavage site. Cleavage of UFD2 by caspases or granzyme B within this putative regulatory N-terminal domain might have important functional consequences within the apoptotic cascade. PMID:11802788

  2. Vertebrate Embryonic Cleavage Pattern Determination.

    PubMed

    Hasley, Andrew; Chavez, Shawn; Danilchik, Michael; Wühr, Martin; Pelegri, Francisco

    2017-01-01

    The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

  3. N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells.

    PubMed

    Kim, Byeong Mo; Choi, Yun Jung; Han, Youngsoo; Yun, Yeon-Sook; Hong, Sung Hee

    2009-08-15

    N,N-dimethyl phytosphingosine (DMPS) blocks the conversion of sphingosine to sphingosine-1-phosphate (S1P) by the enzyme sphingosine kinase (SK). In this study, we elucidated the apoptotic mechanisms of DMPS action on a human leukemia cell line using functional pharmacologic and genetic approaches. First, we demonstrated that DMPS-induced apoptosis is evidenced by nuclear morphological change, distinct internucleosomal DNA fragmentation, and an increased sub-G1 cell population. DMPS treatment led to the activation of caspase-9 and caspase-3, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) and led to cytochrome c release, depolarization of the mitochondrial membrane potential, and downregulation of the anti-apoptotic members of the bcl-2 family. Ectopic expression of bcl-2 and bcl-xL conferred resistance of HL-60 cells to DMPS-induced cell death, suggesting that DMPS-induced apoptosis occurs predominantly through the activation of the intrinsic mitochondrial pathway. We also observed that DMPS activated the caspase-8-Bid-Bax pathway and that the inhibition of caspase-8 by z-IETD-fmk or small interfering RNA suppressed the cleavage of Bid, cytochrome c release, caspase-3 activation, and apoptotic cell death. In addition, cells subjected to DMPS exhibited significantly increased reactive oxygen species (ROS) generation, and ROS scavengers, such as quercetin and Tiron, but not N-acetylcysteine (NAC), inhibited DMPS-induced activations of caspase-8, -3 and subsequent apoptotic cell death, indicating the role of ROS in caspase-8-mediated apoptosis. Taken together, these results indicate that caspase-8 acts upstream of caspase-3, and that the caspase-8-mediated mitochondrial pathway is important in DMPS-induced apoptosis. Our results also suggest that ROS are critical regulators of caspase-8-mediated apoptosis in DMPS-treated leukemia cells.

  4. N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells

    SciTech Connect

    Kim, Byeong Mo; Choi, Yun Jung; Han, Youngsoo; Yun, Yeon-Sook; Hong, Sung Hee

    2009-08-15

    N,N-dimethyl phytosphingosine (DMPS) blocks the conversion of sphingosine to sphingosine-1-phosphate (S1P) by the enzyme sphingosine kinase (SK). In this study, we elucidated the apoptotic mechanisms of DMPS action on a human leukemia cell line using functional pharmacologic and genetic approaches. First, we demonstrated that DMPS-induced apoptosis is evidenced by nuclear morphological change, distinct internucleosomal DNA fragmentation, and an increased sub-G1 cell population. DMPS treatment led to the activation of caspase-9 and caspase-3, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) and led to cytochrome c release, depolarization of the mitochondrial membrane potential, and downregulation of the anti-apoptotic members of the bcl-2 family. Ectopic expression of bcl-2 and bcl-xL conferred resistance of HL-60 cells to DMPS-induced cell death, suggesting that DMPS-induced apoptosis occurs predominantly through the activation of the intrinsic mitochondrial pathway. We also observed that DMPS activated the caspase-8-Bid-Bax pathway and that the inhibition of caspase-8 by z-IETD-fmk or small interfering RNA suppressed the cleavage of Bid, cytochrome c release, caspase-3 activation, and apoptotic cell death. In addition, cells subjected to DMPS exhibited significantly increased reactive oxygen species (ROS) generation, and ROS scavengers, such as quercetin and Tiron, but not N-acetylcysteine (NAC), inhibited DMPS-induced activations of caspase-8, -3 and subsequent apoptotic cell death, indicating the role of ROS in caspase-8-mediated apoptosis. Taken together, these results indicate that caspase-8 acts upstream of caspase-3, and that the caspase-8-mediated mitochondrial pathway is important in DMPS-induced apoptosis. Our results also suggest that ROS are critical regulators of caspase-8-mediated apoptosis in DMPS-treated leukemia cells.

  5. Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis*

    PubMed Central

    Tebbi, Ali; Guittet, Olivier; Tuphile, Karine; Cabrié, Aimeric; Lepoivre, Michel

    2015-01-01

    Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp342 and Asn343. Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis. PMID:25878246

  6. Caspases rule the intracellular trafficking cartel.

    PubMed

    Duclos, Catherine; Lavoie, Christine; Denault, Jean-Bernard

    2017-05-01

    During apoptosis, caspases feast on several hundreds of cellular proteins to orchestrate rapid cellular demise. Indeed, caspases are known to get a taste of every cellular process in one way or another, activating some, but most often shutting them down. Thus, it is not surprising that caspases proteolyze proteins involved in intracellular trafficking with particularly devastating consequences for this important process. This review article focuses on how caspases target the machinery responsible for smuggling goods within and outside the cell. © 2017 Federation of European Biochemical Societies.

  7. TCR-induced, PKC-θ-mediated NF-κB Activation Is Regulated by a Caspase-8-Caspase-9-Caspase-3 Cascade

    PubMed Central

    Zhao, Yixia; Lei, Minxiang; Wang, Zhaoyuan; Qiao, Guilin; Yang, Tianlun; Zhang, Jian

    2014-01-01

    It has been documented that caspase-8, a central player in apoptosis, is also crucial for TCR-mediated NF-κB activation. However, whether other caspases are also involved this process is unknown. In this report, we showed that in addition to caspase-8, caspase-9 is required for TCR-mediated NF-κB activation. Caspase-9 induces activation of PKC-θ, phosphorylation of Bcl10 and NF-κB activation in a caspase-3-dependent manner, but it appears that Bcl10 phosphorylation is uncoupled from NF-κB activation. Furthermore, caspase-8 lies upstream of caspase-9 during T cell activation. Therefore, TCR ligation elicits a caspase cascade involving caspase-8, caspase-9 and caspase-3 which initiates PKC-θ-dependent pathway leading to NF-κB activation and PKC-θ-independent Bcl10 phosphorylation which limits NF-kB activity. PMID:24924627

  8. Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3.

    PubMed

    Karvinen, Jarkko; Hurskainen, Pertti; Gopalakrishnan, Sujatha; Burns, David; Warrior, Usha; Hemmilä, Ilkka

    2002-06-01

    In addition to kinases and G protein-coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/microl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.

  9. Transgenic Mouse Model Expressing the Caspase 6 Fragment of Mutant Huntingtin

    PubMed Central

    Roby, Elaine Waldron; Ratovitski, Tamara; Wang, XiaoFang; Jiang, Mali; Watkin, Erin; Arbez, Nikolas; Graham, Rona K.; Hayden, Michael R.; Hou, Zhipeng; Mori, Susumu; Swing, Deborah; Pletnikov, Mikhail; Duan, Wenzhen; Tessarollo, Lino; Ross, Christopher A.

    2012-01-01

    Huntington’s disease (HD) is caused by a polyglutamine expansion in the Huntingtin (Htt) protein. Proteolytic cleavage of Htt into toxic N-terminal fragments is believed to be a key aspect of pathogenesis. The best characterized putative cleavage event is at amino acid 586, hypothesized to be mediated by caspase 6. A corollary of the caspase 6 cleavage hypothesis is that the caspase 6 fragment should be a toxic fragment. To test this hypothesis, and further characterize the role of this fragment, we have generated transgenic mice expressing the N-terminal 586 aa of Htt with a polyglutamine repeat length of 82 (N586-82Q), under the control of the prion promoter. N586-82Q mice show a clear progressive rotarod deficit by four months of age, and are hyperactive starting at 5 months, later changing to hypoactivity prior to early mortality. MRI studies reveal widespread brain atrophy, and histologic studies demonstrate an abundance of Htt aggregates, mostly cytoplasmic, which are predominantly composed of the N586-82Q polypeptide. Smaller soluble N-terminal fragments appear to accumulate over time, peaking at four months, and are predominantly found in the nuclear fraction. This model appears to have a phenotype more severe than current full-length Htt models, but less severe than HD mouse models expressing shorter Htt fragments. These studies suggest that the caspase 6 fragment may be a transient intermediate, that fragment size is a factor contributing to the rate of disease progression, and that short soluble nuclear fragments may be most relevant to pathogenesis. PMID:22219281

  10. The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles

    PubMed Central

    Orme, Mariam H.; Liccardi, Gianmaria; Moderau, Nina; Feltham, Rebecca; Wicky-John, Sidonie; Tenev, Tencho; Aram, Lior; Wilson, Rebecca; Bianchi, Katiuscia; Morris, Otto; Monteiro Domingues, Celia; Robertson, David; Tare, Meghana; Wepf, Alexander; Williams, David; Bergmann, Andreas; Gstaiger, Matthias; Arama, Eli; Ribeiro, Paulo S.; Meier, Pascal

    2016-01-01

    Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events. PMID:26960254

  11. Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells†

    PubMed Central

    Sosnovtsev, Stanislav V.; Belliot, Gaël; Chang, Kyeong-OK; Prikhodko, Victor G.; Thackray, Larissa B.; Wobus, Christiane E.; Karst, Stephanie M.; Virgin, Herbert W.; Green, Kim Y.

    2006-01-01

    Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication. PMID:16873239

  12. Essential role of caspase-8 in p53/p73-dependent apoptosis induced by etoposide in head and neck carcinoma cells

    PubMed Central

    2011-01-01

    Background Caspase-8 is a key upstream mediator in death receptor-mediated apoptosis and also participates in mitochondria-mediated apoptosis via cleavage of proapoptotic Bid. However, the role of caspase-8 in p53- and p73-dependent apoptosis induced by genotoxic drugs remains unclear. We recently reported that the reconstitution of procaspase-8 is sufficient for sensitizing cisplatin- but not etoposide-induced apoptosis, in chemoresistant and caspase-8 deficient HOC313 head and neck squamous cell carcinoma (HNSCC) cells. Results We show that p53/p73-dependent caspase-8 activation is required for sensitizing etoposide-induced apoptosis by utilizing HOC313 cells carrying a temperature-sensitive p53G285K mutant. Restoration of wild-type p53 function under the permissive conditions, together with etoposide treatment, led to substantial transcriptional activation of proapoptotic Noxa and PUMA, but failed to induce apoptosis. In addition to p53 restoration, caspase-8 reconstitution was needed for sensitization to etoposide-induced apoptosis, mitochondria depolarization, and cleavage of the procaspases-3, and -9. In etoposide-sensitive Ca9-22 cells carrying a temperature-insensitive mutant p53, siRNA-based p73 knockdown blocked etoposide-induced apoptosis and procaspase-8 cleavage. However, induction of p73 protein and up-regulation of Noxa and PUMA, although observed in Ca9-22 cells, were hardly detected in etoposide-treated HOC313 cells under non-permissive conditions, suggesting a contribution of p73 reduction to etoposide resistance in HOC313 cells. Finally, the caspase-9 inhibitor Ac-LEHD-CHO or caspase-9 siRNA blocked etoposide-induced caspase-8 activation, Bid cleavage, and apoptosis in both cell lines, indicating that p53/p73-dependent caspase-8 activation lies downstream of mitochondria. Conclusions we conclude that p53 and p73 can act as upstream regulators of caspase-8, and that caspase-8 is an essential mediator of the p53/p73-dependent apoptosis induced by

  13. RNase L-Independent Specific 28S rRNA Cleavage in Murine Coronavirus-Infected Cells

    PubMed Central

    Banerjee, Sangeeta; An, Sungwhan; Zhou, Aimin; Silverman, Robert H.; Makino, Shinji

    2000-01-01

    We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously. PMID:10982321

  14. Hypoxic induction of caspase-11/caspase-1/interleukin-1beta in brain microglia.

    PubMed

    Kim, Nam-Gon; Lee, Heasuk; Son, Eunyung; Kwon, Oh-Young; Park, Jae-Yong; Park, Jae-Hoon; Cho, Gyeong Jae; Choi, Wan Sung; Suk, Kyoungho

    2003-06-10

    Caspase-11 is an inducible protease that plays an important role in both inflammation and apoptosis. Inflammatory stimuli induce and activate caspase-11, which is required for the activation of caspase-1 or interleukin-1beta (IL-1beta) converting enzyme (ICE). Caspase-1 in turn mediates the maturation of proinflammatory cytokines such as IL-1beta, which is one of the crucial mediators of neurodegeneration in the central nervous system. Here, we report that hypoxic exposure of cultured brain microglia (BV-2 mouse microglia cells and rat primary microglial cultures) induces expression and activation of caspase-11, which is accompanied by activation of caspase-1 and secretion of mature IL-1beta and IL-18. Hypoxic induction of caspase-11 was observed in both mRNA and protein levels, and was mediated through p38 mitogen-activated protein kinase pathway. Transient global ischemia in rats also induced caspase-11 expression and IL-1beta production in hippocampus supporting our in vitro findings. Caspase-11-expressing cells in hippocampus were morphologically identified as microglia. Taken together, our results indicate that hypoxia induces a sequential event-caspase-11 induction, caspase-1 activation, and IL-1beta release-in brain microglia, and point out the importance of initial caspase-11 induction in hypoxia-induced inflammatory activation of microglia.

  15. BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori.

    PubMed

    Yi, Hua-Shan; Pan, Cai-Xia; Pan, Chun; Song, Juan; Hu, Yan-Fen; Wang, La; Pan, Min-Hui; Lu, Cheng

    2014-02-28

    In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a (169)QACRG(173) sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Molecular cloning of sea bass (Dicentrarchus labrax L.) caspase-8 gene and its involvement in Photobacterium damselae ssp. piscicida triggered apoptosis.

    PubMed

    Reis, Marta I R; Costa-Ramos, Carolina; do Vale, Ana; dos Santos, Nuno M S

    2010-07-01

    Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this work, the sea bass counterpart of mammalian caspase-8 was sequenced and characterized, and its involvement in the apoptogenic activity of a toxin from a fish pathogen was assessed. A 2472 bp cDNA of sea bass caspase-8 was obtained, consisting of 1455 bp open reading frame coding for 484 amino acids and with a predicted molecular weight of 55.2 kDa. The sea bass caspase-8 gene has 6639 bp and is organized in 11 introns and 12 exons. Several distinctive features of sea bass caspase-8 were identified, which include two death effector domains, the caspase family domains p20 and p10, the caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. The sea bass caspase-8 sequence revealed a significant degree of similarity to corresponding sequences from several vertebrate taxonomic groups. A low expression of sea bass caspase-8 was detected in various tissues of non-stimulated sea bass. Furthermore, it is shown that stimulation of sea bass with mid-exponential phase culture supernatants from Photobacterium damselae ssp. piscicida (Phdp), known to induce selective apoptosis of macrophages and neutrophils, resulted in an increased expression of caspase-8 in the spleen, one of the main affected organs by Phdp infection.

  17. In Vitro and Ex Vivo Evaluation of Smart Infra-Red Fluorescent Caspase-3 Probes for Molecular Imaging of Cardiovascular Apoptosis

    PubMed Central

    Debunne, Manuelle; Portal, Christophe; Delest, Bruno; Brakenhielm, Ebba; Lallemand, Françoise; Henry, Jean-Paul; Ligeret, Heidi; Noack, Pauline; Massonneau, Marc; Romieu, Anthony; Renard, Pierre-Yves; Thuillez, Christian; Richard, Vincent

    2011-01-01

    Purpose. The aim of this paper is to develop new optical bioprobes for the imaging of apoptosis. Procedure. We developed quenched near-infrared probes which become fluorescent upon cleavage by caspase-3, the key regulatory enzyme of apoptosis. Results. Probes were shown to be selectively cleaved by recombinant caspase-3. Apoptosis of cultured endothelial cells was associated with an increased fluorescent signal for the cleaved probes, which colocalized with caspase-3 and was reduced by the addition of a caspase-3 inhibitor. Flow cytometry demonstrated a similar profile between the cleaved probes and annexin V. Ex vivo experiments showed that sections of hearts obtained from mice treated with the proapoptotic drug doxorubicin displayed an increase in the fluorescent signal for the cleaved probes, which was reduced by a caspase-3 inhibitor. Conclusion. We demonstrated the capacity of these novel probes to detect apoptosis by optical imaging in vitro and ex vivo. PMID:21629849

  18. A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

    PubMed

    Boege, Yannick; Malehmir, Mohsen; Healy, Marc E; Bettermann, Kira; Lorentzen, Anna; Vucur, Mihael; Ahuja, Akshay K; Böhm, Friederike; Mertens, Joachim C; Shimizu, Yutaka; Frick, Lukas; Remouchamps, Caroline; Mutreja, Karun; Kähne, Thilo; Sundaravinayagam, Devakumar; Wolf, Monika J; Rehrauer, Hubert; Koppe, Christiane; Speicher, Tobias; Padrissa-Altés, Susagna; Maire, Renaud; Schattenberg, Jörn M; Jeong, Ju-Seong; Liu, Lei; Zwirner, Stefan; Boger, Regina; Hüser, Norbert; Davis, Roger J; Müllhaupt, Beat; Moch, Holger; Schulze-Bergkamen, Henning; Clavien, Pierre-Alain; Werner, Sabine; Borsig, Lubor; Luther, Sanjiv A; Jost, Philipp J; Weinlich, Ricardo; Unger, Kristian; Behrens, Axel; Hillert, Laura; Dillon, Christopher; Di Virgilio, Michela; Wallach, David; Dejardin, Emmanuel; Zender, Lars; Naumann, Michael; Walczak, Henning; Green, Douglas R; Lopes, Massimo; Lavrik, Inna; Luedde, Tom; Heikenwalder, Mathias; Weber, Achim

    2017-09-11

    Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3

    NASA Astrophysics Data System (ADS)

    Murthy, Aditya; Li, Yun; Peng, Ivan; Reichelt, Mike; Katakam, Anand Kumar; Noubade, Rajkumar; Roose-Girma, Merone; Devoss, Jason; Diehl, Lauri; Graham, Robert R.; van Lookeren Campagne, Menno

    2014-02-01

    Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.

  20. Prion Pathogenesis is Independent of Caspase-12

    PubMed Central

    Steele, Andrew D; Hetz, Claudio; Yi, Caroline H; Jackson, Walker S; Borkowski, Andrew W; Yuan, Junying; Wollmann, Robert H

    2007-01-01

    The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer's disease and many others. The ER stress response and upregulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding. PMID:19164919

  1. Pro-inflammatory caspase-1 activation during the immune response in cells from rainbow trout Oncorhynchus mykiss (Walbaum 1792) challenged with pathogen-associated molecular patterns.

    PubMed

    Rojas, V; Camus-Guerra, H; Guzmán, F; Mercado, L

    2015-11-01

    In response to pathogens, the higher vertebrate innate immune system activates pro-inflammatory caspase-1 which is responsible for the processing and secretion of several important cytokines involved in the host's defence against infection. To date, caspase-1 has been described in few teleost fish, and its activity has been demonstrated through substrate cleavage and inhibition by pharmacological agents. In this study, the detection of the active form of caspase-1 during the immune response in salmonid fish is described, where two antibodies were produced. These antibodies differentially recognize the structural epitopes of the inactive pro-caspase-1 and the processed active form of the caspase. Firstly, caspase-1 activation was demonstrated in vitro by ELISA, Western blotting and immunocytochemistry in rainbow trout macrophages exposed to different pathogen-associated molecular patterns plus the pathogen Aeromonas hydrophila. This activity was clearly abrogated by a caspase inhibitor and seems to be unrelated to IL-1β secretion. Caspase-1 activation was then validated in vivo in gill cells from fish challenged with Aeromonas salmonicida. These results represent the first demonstration of caspase-1 activation in salmonids, and the first evidence of the putative regulatory role which this protease plays in inflammatory response in this fish group, as described for some other teleosts and mammals.

  2. Endoplasmic reticulum stress-induced hepatic stellate cell apoptosis through calcium-mediated JNK/P38 MAPK and Calpain/Caspase-12 pathways.

    PubMed

    Huang, Yan; Li, Xiaohui; Wang, Yarui; Wang, Huan; Huang, Cheng; Li, Jun

    2014-09-01

    Recent reports considered that it was the disturbance of calcium homeostasis and the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that activated hepatic stellate cells (HSCs) apoptosis and promoted fibrosis resolution. However, the signal-transducing events that are activated by ER stress after HSCs activation were incompletely understood. In this study, we induced ER stress with thapsigargin (TG), and determined the activation of calpain and the cleavage of caspase by analyzing the protein levels and the correspondingly increased intracellular calcium levels and the induction of the proapoptotic transcription factor CHOP. Moreover, the phosphorylation of JNK and p38 MAPK were followed by the activation of the executioner caspases, caspase-3. As expected, preventing an increase in intracellular calcium levels using intracellular calcium chelators, EGTA, and BAPTA/AM, could substantially inhibit the phosphorylation of JNK and p38 MAPK, abolish the activation of calpains, namely caspase-12, caspase-9, and caspase-3, and provide significant protection for TG-treated activated HSCs. Interestingly, pretreatment with p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, the pan-caspase inhibitor z-VAD-FMK, or calpain inhibitors calpeptin, significantly reduced the cell apoptosis and the cleavage of caspase-12 and caspase-3. However, pretreatment with z-VAD-FMK failed to reduce the activation of calpain. Additionally, pretreatment with SB202190 and SP600125 also decreased the expression of CHOP. Importantly, PDGF-induced collagen Col1α1 and α-smooth muscle actin (α-SMA), markers for the perpetuation phase of HSCs activation, were inhibited in TG-treated activated HSCs. These findings showed that the Calpain/Caspase-12 activation induced by ER stress and the JNK/p38 MAPK phosphorylation induced by the increase of intracellular calcium concentration releasing from ER are the novel signaling pathway underlying the molecular mechanism of fibrosis

  3. In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity

    PubMed Central

    Tang, Ho Lam; Tang, Ho Man; Fung, Ming Chiu; Hardwick, J. Marie

    2015-01-01

    The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, we developed a dual color CaspaseTracker system for Drosophila to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development. PMID:25757939

  4. Differential roles of caspase-1 and caspase-11 in infection and inflammation

    PubMed Central

    Ming Man, Si; Karki, Rajendra; Briard, Benoit; Burton, Amanda; Gingras, Sebastien; Pelletier, Stephane; Kanneganti, Thirumala-Devi

    2017-01-01

    Caspase-1, also known as interleukin-1β (IL-1β)-converting enzyme (ICE), regulates antimicrobial host defense, tissue repair, tumorigenesis, metabolism and membrane biogenesis. On activation within an inflammasome complex, caspase-1 induces pyroptosis and converts pro-IL-1β and pro-IL-18 into their biologically active forms. “ICE−/−” or “Casp1−/−” mice generated using 129 embryonic stem cells carry a 129-associated inactivating passenger mutation on the caspase-11 locus, essentially making them deficient in both caspase-1 and caspase-11. The overlapping and unique functions of caspase-1 and caspase-11 are difficult to unravel without additional genetic tools. Here, we generated caspase-1–deficient mouse (Casp1Null) on the C57BL/6 J background that expressed caspase-11. Casp1Null cells did not release IL-1β and IL-18 in response to NLRC4 activators Salmonella Typhimurium and flagellin, canonical or non-canonical NLRP3 activators LPS and ATP, Escherichia coli, Citrobacter rodentium and transfection of LPS, AIM2 activators Francisella novicida, mouse cytomegalovirus and DNA, and the infectious agents Listeria monocytogenes and Aspergillus fumigatus. We further demonstrated that caspase-1 and caspase-11 differentially contributed to the host defense against A. fumigatus infection and to endotoxemia. PMID:28345580

  5. Caspase-Cleaved Tau Co-Localizes with Early Tangle Markers in the Human Vascular Dementia Brain

    PubMed Central

    Day, Ryan J.; Mason, Maria J.; Thomas, Chloe; Poon, Wayne W.; Rohn, Troy T.

    2015-01-01

    Vascular dementia (VaD) is the second most common form of dementia in the United States and is characterized as a cerebral vessel vascular disease that leads to ischemic episodes. Whereas the relationship between caspase-cleaved tau and neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD) has been previously described, whether caspase activation and cleavage of tau occurs in VaD is presently unknown. To investigate a potential role for caspase-cleaved tau in VaD, we analyzed seven confirmed cases of VaD by immunohistochemistry utilizing a well-characterized antibody that specifically detects caspase-cleaved tau truncated at Asp421. Application of this antibody (TauC3) revealed consistent labeling within NFTs, dystrophic neurites within plaque-rich regions and corpora amylacea (CA) in the human VaD brain. Labeling of CA by the TauC3 antibody was widespread throughout the hippocampus proper, was significantly higher compared to age matched controls, and co-localized with ubiquitin. Staining of the TauC3 antibody co-localized with MC-1, AT8, and PHF-1 within NFTs. Quantitative analysis indicated that roughly 90% of PHF-1-labeled NFTs contained caspase-cleaved tau. In addition, we documented the presence of active caspase-3 within plaques, blood vessels and pretangle neurons that co-localized with TauC3. Collectively, these data support a role for the activation of caspase-3 and proteolytic cleavage of TauC3 in VaD providing further support for the involvement of this family of proteases in NFT pathology. PMID:26161867

  6. Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender.

    PubMed

    Renolleau, Sylvain; Fau, Sébastien; Goyenvalle, Catherine; Joly, Luc-Marie; Chauvier, David; Jacotot, Etienne; Mariani, Jean; Charriaut-Marlangue, Christiane

    2007-02-01

    Hypoxia-ischaemia in the developing brain results in brain injury with prominent features of apoptosis. In the present study, a third generation dipeptidyl broad-spectrum caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh), was tested in a model of unilateral focal ischaemia with reperfusion in 7-day-old rats. Q-VD-OPh (1 mg/kg, i.p.) reduced cell death, resulting in significant neuroprotection at 48 h of recovery (infarct volume of 12.6 +/- 2.8 vs. 24.3 +/- 2.2%, p = 0.006). The neuroprotective effects observed at 48 h post-ischaemia hold up at 21 days of survival time and attenuate neurological dysfunction. Analysis by gender revealed that females were strongly protected (6.7 +/- 3.3%, p = 0.006), in contrast to males in which there was no significant effect, when Q-VD-OPh was given after clip removal on the left common carotid artery. Immunoblot analysis demonstrated that Q-VD-OPh inhibits caspase 3 cleavage into its p17 active form and caspase 1 up-regulation and cleavage in vivo. Following ischaemia in P7 rats, males and females displayed different time course and pattern of cytochrome c release and active p17 caspase 3 during the first 24 h of recovery. In contrast, no significant difference was observed for caspase 1 expression between genders. These results indicate that ischaemia activates caspases shortly after reperfusion and that the sex of the animal may strongly influences apoptotic pathways in the pathogenesis of neonatal brain injury. The specificity, effectiveness, and reduced toxicity of Q-VD-OPh may determine the potential use of peptide-derived irreversible caspase inhibitors as promising therapeutics.

  7. Apoptosis induction by the glucocorticoid hormone dexamethasone and the calcium-ATPase inhibitor thapsigargin involves Bc1-2 regulated caspase activation.

    PubMed

    McColl, K S; He, H; Zhong, H; Whitacre, C M; Berger, N A; Distelhorst, C W

    1998-04-30

    The requirement for caspases (ICE-like proteases) were investigated in mediating apoptosis of WEHI7.2 mouse lymphoma cells in response to two death inducers with different mechanisms of action, the glucocorticoid hormone dexamethasone (DX) and the calcium-ATPase inhibitor thapsigargin (TG). Apoptosis induction by these agents followed different kinetics, and was closely correlated with in vivo activation of caspase-3 (CPP32/Yama/Apopain) and cleavage of the caspase target protein poly(ADP-ribose) polymerase (PARP). Caspase activation and PARP cleavage were inhibited by Bcl-2 overexpression. Cell extracts from DX- and TG-treated cells cleaved the in vitro synthesized baculovirus p35 ICE-like protease target, producing 25 and 10 kDa fragments. p35 cleavage was inhibited by mutating the active site aspartic acid to alanine, and by a panel of protease inhibitors that inhibit caspase-3-like proteases, including iodoacetamide, N-ethylmaleimide, and Ac-DEVD-cho. Treatment of cells in vivo with two cell permeant peptide fluoromethylketone inhibitors of caspase activity, Z-VAD-fmk and Z-DEVD-fmk, inhibited DX- and TG-induced apoptotic nuclear changes and maintained plasma membrane integrity, whereas the cathepsin inhibitor, Z-FA-fmk, and two calpain inhibitors failed to inhibit apoptosis. An unexpected observation was that due to the delayed time course of DX-induced apoptosis, optimal preservation of plasma membrane integrity was achieved by adding caspase inhibitors beginning 8 h after DX addition. In summary, the findings indicate that two diverse apoptosis-inducing signals converge into a common Bcl-2-regulated pathway that leads to caspase activation and apoptosis.

  8. Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis.

    PubMed Central

    Rodriguez-Lafrasse, C; Alphonse, G; Broquet, P; Aloy, M T; Louisot, P; Rousson, R

    2001-01-01

    To clarify the chronology of events leading to anti-Fas-induced apoptosis, and the mechanisms of resistance to this death effector, we compared the response kinetics of three tumour cell lines that display varying sensitivity to anti-Fas (based on levels of apoptosis), in terms of ceramide release, mitochondrial function and the caspase-activation pathway. In the highly sensitive Jurkat cell line, early caspase-8 activation, observed from 2 h after treatment, was chronologically associated with an acute depletion of glutathione and the cleavage of caspase-3 and poly-ADP ribosyl polymerase (PARP), followed by a progressive fall in the mitochondrial transmembrane potential (Delta(psi)m), between 4 and 48 h after treatment. Ceramide levels began to increase 2 h after the addition of anti-Fas (with no increase during the first hour), and increased continuously to 640% of control cells at 48 h. In the moderately sensitive SCC61 adherent cells, comparable results were observed, though with lower levels of ceramide and a delay in the response kinetics, with apoptotic cells becoming flotant. Finally, despite early cleavage of caspase-8 at 2 h, and a sustained level of activation until 48 h, no apoptotic response was observed in anti-Fas-resistant SQ20B cells. This was confirmed by a lack of ceramide generation and mitochondrial changes, and by the absence of any detectable cleavage of caspase-3 or PARP. Inhibition of caspase processing, and amplification of endogenous ceramide signalling by pharmacological agents, allowed us to establish the order of cellular events, locating ceramide release after caspase-8 activation and before caspase-3 activation, and demonstrating a direct involvement for ceramide release in mitochondrial dysfunction. Furthermore, these experiments provide strong arguments for the role of endogenous ceramide as a key executor of apoptosis, rather than as a consequence of membrane alterations. PMID:11439090

  9. Regulation of CED-3 caspase localization and activation by C. elegans nuclear membrane protein NPP-14

    PubMed Central

    Chen, Xudong; Wang, Yue; Chen, Yu-Zen; Harry, Brian L.; Nakagawa, Akihisa; Lee, Eui-Seung; Guo, Hongyan; Xue, Ding

    2017-01-01

    Caspases are cysteine proteases with critical roles in apoptosis. The Caenorhabditis elegans caspase CED-3 is activated by autocatalytic cleavage, a process enhanced by CED-4. Here we report that CED-3 zymogen localizes to the perinuclear region in C. elegans germ cells and that CED-3 autocatalytic cleavage is held in check by C. elegans nuclei and activated by CED-4. The nuclear pore protein NPP-14 interacts with the prodomain of CED-3 zymogen, colocalizes with CED-3 in vivo, and inhibits CED-3 autoactivation in vitro. Several missense mutations in the CED-3 prodomain result in stronger association with NPP-14 and reduced CED-3 activation by CED-4 in the presence of nuclei or NPP-14, leading to cell death defects. Those same mutations enhance autocatalytic cleavage of CED-3 in vitro and increase apoptosis in vivo in the absence of npp-14. Our results reveal a critical role for nuclei and nuclear membrane proteins in regulating activation and localization of CED-3. PMID:27723735

  10. Caspase-mediated proteolysis of the sorting nexin 2 disrupts retromer assembly and potentiates Met/hepatocyte growth factor receptor signaling

    PubMed Central

    Duclos, Catherine M; Champagne, Audrey; Carrier, Julie C; Saucier, Caroline; Lavoie, Christine L; Denault, Jean-Bernard

    2017-01-01

    The unfolding of apoptosis involves the cleavage of hundreds of proteins by the caspase family of cysteinyl peptidases. Among those substrates are proteins involved in intracellular vesicle trafficking with a net outcome of shutting down the crucial processes governing protein transport to organelles and to the plasma membrane. However, because of the intertwining of receptor trafficking and signaling, cleavage of specific proteins may lead to unintended consequences. Here we show that in apoptosis, sorting nexin 1 and 2 (SNX1 and SNX2), two proteins involved in endosomal sorting, are cleaved by initiator caspases and also by executioner caspase-6 in the case of SNX2. Moreover, SNX1 is cleaved at multiple sites, including following glutamate residues. Cleavage of SNX2 results in a loss of association with the endosome-to-trans-Golgi network transport protein Vps35 and in a delocalization from endosomes of its associated partner Vps26. We also demonstrate that SNX2 depletion causes an increase in hepatocyte growth factor receptor tyrosine phosphorylation and Erk1/2 signaling in cells. Finally, we show that SNX2 mRNA and protein levels are decreased in colorectal carcinoma and that lower SNX2 gene expression correlates with an increase in cancer patient mortality. Our study reveals the importance to characterize the cleavage fragments produced by caspases of specific death substrates given their potential implication in the mechanism of regulation of physiological (signaling/trafficking) pathways or in the dysfunction leading to pathogenesis. PMID:28179995

  11. E1A enhances cellular sensitivity to DNA-damage-induced apoptosis through PIDD-dependent caspase-2 activation.

    PubMed

    Radke, Jay R; Siddiqui, Zeba K; Figueroa, Iris; Cook, James L

    Expression of the adenoviral protein, E1A, sensitizes mammalian cells to a wide variety of apoptosis-inducing agents through multiple cellular pathways. For example, E1A sensitizes cells to apoptosis induced by TNF-superfamily members by inhibiting NF-kappa B (NF-κB)-dependent gene expression. In contrast, E1A sensitization to nitric oxide, an inducer of the intrinsic apoptotic pathway, is not dependent upon repression of NF-κB-dependent transcription but rather is dependent upon caspase-2 activation. The latter observation suggested that E1A-induced enhancement of caspase-2 activation might be a critical factor in cellular sensitization to other intrinsic apoptosis pathway-inducing agents. Etoposide and gemcitabine are two DNA damaging agents that induce intrinsic apoptosis. Here we report that E1A-induced sensitization to both of these agents, like NO, is independent of NF-κB activation but dependent on caspase-2 activation. The results show that caspase-2 is a key mitochondrial-injuring caspase during etoposide and gemcitabine-induced apoptosis of E1A-positive cells, and that caspase-2 is required for induction of caspase-3 activity by both chemotherapeutic agents. Expression of PIDD was required for caspase-2 activation, mitochondrial injury and enhanced apoptotic cell death. Furthermore, E1A-enhanced sensitivity to injury-induced apoptosis required PIDD cleavage to PIDD-CC. These results define the PIDD/caspase-2 pathway as a key apical, mitochondrial-injuring mechanism in E1A-induced sensitivity of mammalian cells to chemotherapeutic agents.

  12. E1A enhances cellular sensitivity to DNA-damage-induced apoptosis through PIDD-dependent caspase-2 activation

    PubMed Central

    Radke, Jay R; Siddiqui, Zeba K; Figueroa, Iris; Cook, James L

    2016-01-01

    Expression of the adenoviral protein, E1A, sensitizes mammalian cells to a wide variety of apoptosis-inducing agents through multiple cellular pathways. For example, E1A sensitizes cells to apoptosis induced by TNF-superfamily members by inhibiting NF-kappa B (NF-κB)-dependent gene expression. In contrast, E1A sensitization to nitric oxide, an inducer of the intrinsic apoptotic pathway, is not dependent upon repression of NF-κB-dependent transcription but rather is dependent upon caspase-2 activation. The latter observation suggested that E1A-induced enhancement of caspase-2 activation might be a critical factor in cellular sensitization to other intrinsic apoptosis pathway-inducing agents. Etoposide and gemcitabine are two DNA damaging agents that induce intrinsic apoptosis. Here we report that E1A-induced sensitization to both of these agents, like NO, is independent of NF-κB activation but dependent on caspase-2 activation. The results show that caspase-2 is a key mitochondrial-injuring caspase during etoposide and gemcitabine-induced apoptosis of E1A-positive cells, and that caspase-2 is required for induction of caspase-3 activity by both chemotherapeutic agents. Expression of PIDD was required for caspase-2 activation, mitochondrial injury and enhanced apoptotic cell death. Furthermore, E1A-enhanced sensitivity to injury-induced apoptosis required PIDD cleavage to PIDD-CC. These results define the PIDD/caspase-2 pathway as a key apical, mitochondrial-injuring mechanism in E1A-induced sensitivity of mammalian cells to chemotherapeutic agents. PMID:27833761

  13. A designed redox-controlled caspase

    SciTech Connect

    Witkowski, Witold A.; Hardy, Jeanne A.

    2011-09-15

    Caspases are a powerful class of cysteine proteases. Introduction of activated caspases in healthy or cancerous cells results in induction of apoptotic cell death. In this study, we have designed and characterized a version of caspase-7 that can be inactivated under oxidizing extracellular conditions and then reactivated under reducing intracellular conditions. This version of caspase-7 is allosterically inactivated when two of the substrate-binding loops are locked together via an engineered disulfide. When this disulfide is reduced, the protein regains its full function. The inactive loop-locked version of caspase-7 can be readily observed by immunoblotting and mass spectrometry. The reduced and reactivated form of the enzyme observed crystallographically is the first caspase-7 structure in which the substrate-binding groove is properly ordered even in the absence of an active-site ligand. In the reactivated structure, the catalytic-dyad cysteine-histidine are positioned 3.5 {angstrom} apart in an orientation that is capable of supporting catalysis. This redox-controlled version of caspase-7 is particularly well suited for targeted cell death in concert with redox-triggered delivery vehicles.

  14. Caspases and their role in gastric cancer.

    PubMed

    Frejlich, Ewelina; Rudno-Rudzińska, Julia; Janiszewski, Kacper; Salomon, Lukasz; Kotulski, Krzysztof; Pelzer, Oskar; Grzebieniak, Zygmunt; Tarnawa, Robert; Kielan, Wojciech

    2013-01-01

    Caspases (Cysteine Aspartate Specific Proteases) are a group of cysteine-containing proteolytic enzymes produced by the cells of living organisms. They participate in immunological functions, proliferation, cell migration and organization. Caspases also influence the secretion of various regulative factors. Moreover, they are responsible for cellular maturation and reconstruction, and for regulating the number and quality of cells initiating the apoptosis of old cells or those that cannot play their normal role due to abnormalities. Multiple pathological processes are associated with disorders in the activity of caspases. Changes in expression of individual caspases have been observed in gastric cancer. The expression of some caspases is also correlated with particular histological traits and the frequency of metastases, which suggests their possible use as a prognostic factor. It has also been discovered that some somatic mutations in caspase coding genes might lead to inhibition of apoptosis and the progression of the disease. Gene polymorphism may be a gastric cancer risk factor, but may also play a protective function. Considering the less than satisfactory effects of conventional therapeutic methods, the search for alternative ways to activate apoptosis - through gene therapy or selective activation of individual elements of the apoptotic pathways - constitutes a promising direction for studies of new therapeutic strategies. Caspases, enzymes playing a central role in the process of programmed cellular death, may possibly be a key to the development of a more effective anti-cancer therapy.

  15. A novel cell lysis approach reveals that caspase-2 rapidly translocates from the nucleus to the cytoplasm in response to apoptotic stimuli.

    PubMed

    Tinnikov, Alexander A; Samuels, Herbert H

    2013-01-01

    Unlike other caspases, caspase-2 appears to be a nuclear protein although immunocytochemical studies have suggested that it may also be localized to the cytosol and golgi. Where and how caspase-2 is activated in response to apoptotic signals is not clear. Earlier immunocytochemistry studies suggest that caspase-2 is activated in the nucleus and through cleavage of BID leads to increased mitochondrial permeability. More recent studies using bimolecular fluorescence complementation found that caspase-2 oligomerization that leads to activation only occurs in the cytoplasm. Thus, apoptotic signals may lead to activation of caspase-2 which may already reside in the cytoplasm or lead to release of nuclear caspase-2 to the extra-nuclear cytoplasmic compartment. It has not been possible to study release of nuclear caspase-2 to the cytoplasm by cell fractionation studies since cell lysis is known to release nuclear caspase-2 to the extra-nuclear fraction. This is similar to what is known about unliganded nuclear estrogen receptor-α (ERα ) when cells are disrupted. In this study we found that pre-treatment of cells with N-ethylmaleimide (NEM), which alkylates cysteine thiol groups in proteins, completely prevents redistribution of caspase-2 and ERα from the nucleus to the extra-nuclear fraction when cells are lysed. Using this approach we provide evidence that apoptotic signals rapidly leads to a shift of caspase-2 from the nucleus to the extra-nuclear fraction, which precedes the detection of apoptosis. These findings are consistent with a model where apoptotic signals lead to a rapid shift of caspase-2 from the nucleus to the cytoplasm where activation occurs.

  16. Caspase-14: A novel caspase in the retina with a potential role in diabetic retinopathy

    PubMed Central

    Ahmad, Saif; Megyerdi, Sylvia; Othman, Amira; Baban, Babak; Palenski, Tammy L.; Shin, Eui Seok; Gurel, Zafer; Hsu, Stephen; Sheibani, Nader

    2012-01-01

    Purpose The purpose of this study was to evaluate caspase-14 expression in the retina under normal and diabetic conditions, and to determine whether caspase-14 contributes to retinal microvascular cell death under high glucose conditions. Methods Quantitative real-time polymerase chain reaction and western blot analysis were used to evaluate caspase-14 expression in retinal cells, including pericytes (PCs), endothelial cells (ECs), astrocytes (ACs), choroidal ECs, and retinal pigment epithelium (RPE) cells. We also determined caspase-14 expression in the retinas of human subjects with or without diabetic retinopathy (DR) and in experimental diabetic mice. Retinal ECs and PCs were infected with adenoviruses expressing human caspase-14 or green fluorescent protein. Caspase-14 expression was also assessed in retinal vascular cells cultured under high glucose conditions. The number of apoptotic cells was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling staining and confirmed by determining the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3. Results Our experiments demonstrated that retinal ECs, PCs, ACs, choroidal ECs, and RPE cells expressed caspase-14, and DR was associated with upregulation and/or activation of caspase-14 particularly in retinal vasculature. High glucose induced marked elevation of the caspase-14 level in retinal vascular cells. There was a significant increase in the apoptosis rate and the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3 in retinal ECs and PCs overexpressing caspase-14. Conclusions Our findings indicate that caspase-14 might play a significant role in the pathogenesis of DR by accelerating retinal PC and EC death. Further investigations are required to elaborate the underlying mechanisms. PMID:22876114

  17. Activation of Bcl-2-Caspase-9 Apoptosis Pathway in the Testis of Asthmatic Mice

    PubMed Central

    Li, Junjuan; Ding, Zhaolei; Sheng, Jianhui; Li, Juan; Tan, Wei

    2016-01-01

    Background Apoptosis plays a critical role in controlling the proliferation and differentiation of germ cells during spermatogenesis. Dysregulation of the fine-tuned balance may lead to the onset of testicular diseases. In this study, we investigated the activation status of apoptosis pathways in the testicular tissues under the background of an asthmatic mouse model. Methods Ten BALB/c mice were divided into two groups: the acute asthma group and the control group. In the acute asthma group, ovalbumin (OVA)-sensitized mice were challenged with aerosolized OVA for 7 days, while the control group was treated with physiological saline. After that, both epididymis and testis were collected to determine the sperm count and motility. Apoptosis in the testis was evaluated by DNA ladder, immunochemistry and further by PCR array of apoptosis-related genes. Finally, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP) was determined by western blot and the enzymatic activities of caspase-9 and 3/7 were assessed using Caspase-Glo kits. Results Compared with control mice, significant decreases in the body weight, testis weight, sperm count and motility were seen in the experimental group. DNA ladder and immunochemistry showed significant increase in apoptotic index of the asthmatic testis, whereas a decrease in mRNA expression of Bcl-2 and increases in Bax, BNIP3, caspase-9, and AIF were observed in the asthma group. Furthermore, protein levels of AIF were significantly upregulated, while the translational expression of Bcl-2 was downregulated markedly. Consistently, caspase-9 activity in the testis of asthma mice was significantly higher than that of the control group. Conclusion Collectively, these results showed that Bcl-2-caspase-9 apoptosis pathway was clearly activated in the testis of asthmatic mice with the increased expression of apoptosis-related genes and proteins. To our knowledge, this is the first report demonstrating that asthma could lead to the

  18. Activation of Bcl-2-Caspase-9 Apoptosis Pathway in the Testis of Asthmatic Mice.

    PubMed

    Xu, Wenyuan; Guo, Guifang; Li, Junjuan; Ding, Zhaolei; Sheng, Jianhui; Li, Juan; Tan, Wei

    2016-01-01

    Apoptosis plays a critical role in controlling the proliferation and differentiation of germ cells during spermatogenesis. Dysregulation of the fine-tuned balance may lead to the onset of testicular diseases. In this study, we investigated the activation status of apoptosis pathways in the testicular tissues under the background of an asthmatic mouse model. Ten BALB/c mice were divided into two groups: the acute asthma group and the control group. In the acute asthma group, ovalbumin (OVA)-sensitized mice were challenged with aerosolized OVA for 7 days, while the control group was treated with physiological saline. After that, both epididymis and testis were collected to determine the sperm count and motility. Apoptosis in the testis was evaluated by DNA ladder, immunochemistry and further by PCR array of apoptosis-related genes. Finally, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP) was determined by western blot and the enzymatic activities of caspase-9 and 3/7 were assessed using Caspase-Glo kits. Compared with control mice, significant decreases in the body weight, testis weight, sperm count and motility were seen in the experimental group. DNA ladder and immunochemistry showed significant increase in apoptotic index of the asthmatic testis, whereas a decrease in mRNA expression of Bcl-2 and increases in Bax, BNIP3, caspase-9, and AIF were observed in the asthma group. Furthermore, protein levels of AIF were significantly upregulated, while the translational expression of Bcl-2 was downregulated markedly. Consistently, caspase-9 activity in the testis of asthma mice was significantly higher than that of the control group. Collectively, these results showed that Bcl-2-caspase-9 apoptosis pathway was clearly activated in the testis of asthmatic mice with the increased expression of apoptosis-related genes and proteins. To our knowledge, this is the first report demonstrating that asthma could lead to the activation of the mitochondrial apoptosis

  19. BmDredd is an initiator caspase and participates in Emodin-induced apoptosis in the silkworm, Bombyx mori.

    PubMed

    Wang, La; Song, Juan; Bao, Xi-Yan; Chen, Peng; Yi, Hua-Shan; Pan, Min-Hui; Lu, Cheng

    2016-10-15

    The identification and analysis of the caspases is essential to research into apoptosis in lepidoptera insects. The domesticated silkworm, Bombyx mori, is the model system for lepidopterans. In this study, we cloned and characterized a B. mori Dredd gene, BmDredd, the proposed insect homologue of human caspase-8, which encoded a polypeptide of 543 amino acids. BmDredd possesses a long N-terminal prodomain, a p20 domain, and a p10 domain. When transiently expressed in Escherichia coli cells, BmDredd underwent spontaneous cleavage and exhibited high proteolytic activity for caspase-8 substrate but relatively low for caspase-3 or -9 substrate. In addition, BmDredd induced apoptosis when transiently expressed in BmN-SWU1 cells, an ovarian cell line of B. mori. Moreover, after the treatment of Emodin, a novel apoptosis inducer, endogenous BmDredd expression level, the caspase-8 activity and the apoptotic rate increased notably in BmN-SWU1 cells. When BmDredd was subjected to interference in BmN-SWU1 cells and Emodin treatment, BmDredd expression levels decreased and the apoptotic rate also decreased significantly. These results suggest BmDredd is the homologue of human caspase-8 and plays a role in Emodin-induced apoptosis in BmN-SWU1 cells of B. mori. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Apigenin Induces Apoptosis through a Mitochondria/Caspase-Pathway in Human Breast Cancer MDA-MB-453 Cells.

    PubMed

    Choi, Eun Jeong; Kim, Gun-Hee

    2009-05-01

    In this study, we investigated the mechanistic role of the caspase cascade in extrinsic and intrinsic apoptosis induced by apigenin, which has been targeted as a candidate in the development of noncytotoxic anticancer medicines. Treatment with apigenin (1-100 microM) significantly inhibited the proliferation of MDA-MB-453 human breast cancer cells in a dose- and time-dependent manner with IC(50) values of 59.44 and 35.15 microM at 24 and 72 h, respectively. This inhibition resulted in the induction of apoptosis and the release of cytochrome c in cells exposed to apigenin at its 72 h IC(50). Subsequently, caspase-9, which acts in mitochondria-mediated apoptosis, was cleaved by apigenin. In addition, apigenin activated caspase-3, which functions downstream of caspase-9. The apigenin-induced activation of caspase-3 was accompanied by the cleavage of capases-6, -7, and -8. These results are supported by evidence showing that the activity patterns of caspases-3, -8, and -9 were similar. The present study supports the hypothesis that apigenin-induced apoptosis involves the activation of both the intrinsic and extrinsic apoptotic pathways.

  1. Desferrioxamine (DFX) induces apoptosis through the p38-caspase8-Bid-Bax pathway in PHA-stimulated human lymphocytes

    SciTech Connect

    Kim, Byeong-Mo; Chung, Hai-Won

    2008-04-01

    Desferrioxamine (DFX) induces apoptosis in human lymphocytes, although the mechanism leading to cell death is unclear. Therefore, we investigated the signaling pathways implicated in DFX-induced apoptosis in lymphocytes. DFX treatment activated caspase-9, caspase-3, and caspase-8. DFX-induced apoptosis was inhibited by both z-IETD-fmk and z-DEVD-fmk. DFX treatment also enhanced caspase-8 activity, Bid cleavage, and the conformational activation of Bax. DFX treatment activated two MAPKs, p38 and JNK, and induced the phosphorylation of two proteins in the p38 pathway, MKK3 and MKK6. DFX treatment also increased the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2, indicating that DFX promotes p38 activity. In addition, the selective p38 inhibitor SB203580 suppressed DFX-induced apoptosis and caspase-8 activation, whereas the JNK inhibitor, SP600125, and the ERK inhibitor, PD98059, had no effect. Our results suggest that DFX-induced apoptosis is mediated by the p38 pathway and a caspase-8-dependent Bid-Bax pathway in human lymphocytes.

  2. Lantana camara Induces Apoptosis by Bcl-2 Family and Caspases Activation.

    PubMed

    Han, Eun Byeol; Chang, Bo Yoon; Jung, Young Suk; Kim, Sung Yeon

    2015-04-01

    Breast cancer is one of the most common cancers worldwide, and the second most fatal cancer in women after lung cancer. Because there are instances of cancer resistance to existing therapies, studies focused on the identification of novel therapeutic drugs are very important. In this study, we identified a natural anticancer agent from Lantana camara, a flowering plant species of the genus Verbena. The extract obtained from the L. camara exhibited cell death properties in the human breast cancer cell line, MCF-7. We found that the apoptosis induced by treatment with the L. camara extract was regulated by the Bcl-2 family. Bid and Bax was increased and Bcl-2 was decreased by L. camara extract. L. camara extract modulated cleavage of caspase-8, and caspase-9, as well as poly (ADP-ribose) polymerase (PARP). Our results support the potential use of the L. camara extract as an anti-breast cancer drug.

  3. Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

    PubMed Central

    Zorman, Jernej; Sušjan, Petra; Hafner-Bratkovič, Iva

    2016-01-01

    Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions. PMID:27467658

  4. TAK1 regulates caspase 8 activation and necroptotic signaling via multiple cell death checkpoints

    PubMed Central

    Guo, Xiaoyun; Yin, Haifeng; Chen, Yi; Li, Lei; Li, Jing; Liu, Qinghang

    2016-01-01

    Necroptosis has emerged as a new form of programmed cell death implicated in a number of pathological conditions such as ischemic injury, neurodegenerative disease, and viral infection. Recent studies indicate that TGFβ-activated kinase 1 (TAK1) is nodal regulator of necroptotic cell death, although the underlying molecular regulatory mechanisms are not well defined. Here we reported that TAK1 regulates necroptotic signaling as well as caspase 8-mediated apoptotic signaling through both NFκB-dependent and -independent mechanisms. Inhibition of TAK1 promoted TNFα-induced cell death through the induction of RIP1 phosphorylation/activation and necrosome formation. Further, inhibition of TAK1 triggered two caspase 8 activation pathways through the induction of RIP1-FADD-caspase 8 complex as well as FLIP cleavage/degradation. Mechanistically, our data uncovered an essential role for the adaptor protein TNF receptor-associated protein with death domain (TRADD) in caspase 8 activation and necrosome formation triggered by TAK1 inhibition. Moreover, ablation of the deubiqutinase CYLD prevented both apoptotic and necroptotic signaling induced by TAK1 inhibition. Finally, blocking the ubiquitin-proteasome pathway prevented the degradation of key pro-survival signaling proteins and necrosome formation. Thus, we identified new regulatory mechanisms underlying the critical role of TAK1 in cell survival through regulation of multiple cell death checkpoints. Targeting key components of the necroptotic pathway (e.g., TRADD and CYLD) and the ubiquitin-proteasome pathway may represent novel therapeutic strategies for pathological conditions driven by necroptosis. PMID:27685625

  5. Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1.

    PubMed

    Zorman, Jernej; Sušjan, Petra; Hafner-Bratkovič, Iva

    2016-01-01

    Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.

  6. Hyperosmotic Stress Induces Tau Proteolysis by Caspase-3 Activation in SH-SY5Y Cells.

    PubMed

    Olivera-Santa Catalina, Marta; Caballero-Bermejo, Montaña; Argent, Ricardo; Alonso, Juan C; Cuenda, Ana; Lorenzo, María J; Centeno, Francisco

    2016-12-01

    Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of μ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor μ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. J. Cell. Biochem. 117: 2781-2790, 2016. © 2016 Wiley Periodicals, Inc.

  7. Caspase-11 Requires the Pannexin-1 Channel and the Purinergic P2X7 Pore to Mediate Pyroptosis and Endotoxic Shock.

    PubMed

    Yang, Dahai; He, Yuan; Muñoz-Planillo, Raul; Liu, Qin; Núñez, Gabriel

    2015-11-17

    The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis, which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel followed up by ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by cytosolic LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or Toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11(-/-), Panx1(-/-), or P2x7(-/-) mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Caspase-11 requires the pannexin-1 channel and the purinergic P2X7 pore to mediate pyroptosis and endotoxic shock

    PubMed Central

    Yang, Dahai; He, Yuan; Muñoz-Planillo, Raul; Liu, Qin; Núñez, Gabriel

    2016-01-01

    SUMMARY The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel and ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by LPS transfection or treatment with cholera toxin B and LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11−/−, Panx1−/− or P2x7−/− mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome. PMID:26572062

  9. Function of caspase-14 in trophoblast differentiation

    PubMed Central

    White, Lloyd J; Declercq, Wim; Arfuso, Frank; Charles, Adrian K; Dharmarajan, Arun M

    2009-01-01

    Background Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored. Methods Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways. Results Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted. Conclusion Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease. PMID:19747408

  10. ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication.

    PubMed

    Kitazawa, Masato; Hida, Shigeaki; Fujii, Chifumi; Taniguchi, Shun'ichiro; Ito, Kensuke; Matsumura, Tomio; Okada, Nagisa; Sakaizawa, Takashi; Kobayashi, Akira; Takeoka, Michiko; Miyagawa, Shin-Ichi

    2017-01-01

    ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy.

  11. Interaction between caspase-8 activation and endoplasmic reticulum stress in glycochenodeoxycholic acid-induced apoptotic HepG2 cells.

    PubMed

    Iizaka, Toru; Tsuji, Mayumi; Oyamada, Hideto; Morio, Yuri; Oguchi, Katsuji

    2007-11-30

    The accumulation of hydrophobic bile acid, such as glycochenodeoxycholic acid (GCDCA), in the liver has been thought to induce hepatocellular damage in human chronic cholestatic liver diseases. We previously reported that GCDCA-induced apoptosis was promoted by both mitochondria-mediated and endoplasmic reticulum (ER) stress-associated pathways in rat hepatocytes. In this study, we elucidated the relationship between these pathways in GCDCA-induced apoptotic HepG2 cells. HepG2 cells were treated with GCDCA (100-500microM) with or without a caspase-8 inhibitor, Z-IETD-fluoromethyl ketone (Z-IETD-FMK) (30microM) for 3-24h. We demonstrated the presence of both apoptotic pathways in these cells; that is, we showed increases in cleaved caspase-3 proteins, the release of cytochrome c from mitochondria, and the expression of ER resident molecular chaperone Bip mRNA and ER stress response-associated transcription factor Chop mRNA. On the other hand, pretreatment with Z-IETD-FMK significantly reduced the increases, compared with treatment with GCDCA alone. Immunofluorescence microscopic analysis showed that treatment with GCDCA increased the cleavage of BAP31, an integral membrane protein of ER, and pretreatment with Z-IETD-FMK suppressed the increase of caspase-8 and BAP31 cleavage. In conclusion, these results suggest that intact activated caspase-8 may promote and amplify the ER stress response by cleaving BAP31 in GCDCA-induced apoptotic cells.

  12. Analysis of caspase-3 in ASTC-a-1 cells treated with mitomycin C using acceptor photobleaching techniques

    NASA Astrophysics Data System (ADS)

    Wang, Huiying; Chen, Tongsheng; Sun, Lei

    2008-02-01

    Caspase-3 is a key activated death protease, which catalyzes the specific cleavage of many cellular proteins and induces DNA cleavage eventually. In this report, cells were treated with mitomycin C (MMC) at different concentration and its activity was detected by cell counting kit (CCK-8). Based on results of CCK-8, cells were treated with 10μg/mL MMC and Hoechst 33258 has been used to observe cell apoptosis. Fluorescence resonance energy transfer (FRET) and confocal microscopy have been used to the effect of MMC on the caspase3 activation in living cells. Human lung adenocarcinoma cells (ASTC-a-1) was transfected with plasmid SCAT3 (pSCAT3)/CKAR FRET receptor. Acceptor photobleaching techniques of FRET plasmid has been used to destruct fluorophore of cells stably expressing SCAT3 reporter on a fluorescence confocal microscope. The activity of caspase3 can be analyzed by FRET dynamics of SCAT3 in living cells. Our results show that MM C can induce ASTC-a-1 cell apoptosis through activation of caspase3.

  13. Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

    PubMed Central

    Caution, Kyle; Gavrilin, Mikhail A.; Tazi, Mia; Kanneganti, Apurva; Layman, Daniel; Hoque, Sheshadri; Krause, Kathrin; Amer, Amal O.

    2015-01-01

    Inflammasomes are multiprotein complexes that include members of the NOD-like receptor family and caspase-1. Caspase-1 is required for the fusion of the Legionella vacuole with lysosomes. Caspase-11, independently of the inflammasome, also promotes phagolysosomal fusion. However, it is unclear how these proteases alter intracellular trafficking. Here, we show that caspase-11 and caspase-1 function in opposing manners to phosphorylate and dephosphorylate cofilin, respectively upon infection with Legionella. Caspase-11 targets cofilin via the RhoA GTPase, whereas caspase-1 engages the Slingshot phosphatase. The absence of either caspase-11 or caspase-1 maintains actin in the polymerized or depolymerized form, respectively and averts the fusion of pathogen-containing vacuoles with lysosomes. Therefore, caspase-11 and caspase-1 converge on the actin machinery with opposing effects to promote vesicular trafficking. PMID:26686473

  14. Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway.

    PubMed

    Lin, Meng-Liang; Lu, Yao-Cheng; Chung, Jing-Gung; Li, Yi-Chen; Wang, Shyang-Guang; N G, Sue-Hwee; Wu, Chia-Yin; Su, Hong-Lin; Chen, Shih-Shun

    2010-05-01

    Aloe-emodin (AE), a natural, biologically active compound from the rhizome of Rheum palmatum, has been shown to induce apoptosis in several cancer cell lines in vitro. However, its molecular mechanism of action in the apoptosis induction of human nasopharyngeal carcinoma (NPC) cells has not been explored. This study shows that AE induced G(2)/M phase arrest by increasing levels of cyclin B1 bound to Cdc2, and also caused an increase in apoptosis of NPC cells, which was characterized by morphological changes, nuclear condensation, DNA fragmentation, caspase-3 activation, cleavage of poly (ADP-ribose) polymerase (PARP) and increased sub-G(1) population. Treatment of NPC cells with AE also resulted in a decrease in Bcl-X(L) and an increase in Bax expression. Ectopic expression of Bcl-X(L) but not Bcl-2 or small interfering RNA (siRNA)-mediated attenuation of Bax suppressed AE-induced apoptotic cell death. AE-induced loss of mitochondrial membrane potential (MMP) and increase in cellular Ca(++) content, reactive oxygen species (ROS) and apoptotic cell death were suppressed by the treatment of cyclosporin A (CsA) or caspase-8 inhibitor Z-IETD-FMK. Co-treatment with caspase-9 inhibitor Z-LEHD-FMK could inhibit AE-induced cell death and the activation of caspase-3 and -9. In addition, suppression of caspase-8 with the specific inhibitor Z-IETD-FMK inhibited AE-induced the activation of Bax, the cleavage of Bid, the translocation of tBid to the mitochondria and the release of cytochrome c, apoptosis-inducing factor (AIF) and Endo G from the mitochondria and subsequent apoptosis. Taken together, these results indicate that the caspase-8-mediated activation of the mitochondrial death pathway plays a critical role in AE-induced apoptosis of NPC cells.

  15. DNA-ligase IV and DNA-protein kinase play a critical role in deficient caspases activation in apoptosis-resistant cancer cells by using doxorubicin.

    PubMed

    Friesen, Claudia; Uhl, Miriam; Pannicke, Ulrich; Schwarz, Klaus; Miltner, Erich; Debatin, Klaus-Michael

    2008-08-01

    Resistance toward cytotoxic drugs is one of the primary causes for therapeutic failure in cancer therapy. DNA repair mechanisms as well as deficient caspases activation play a critical role in apoptosis resistance of tumor cells toward anticancer drug treatment. Here, we discovered that deficient caspases activation in apoptosis-resistant cancer cells depends on DNA-ligase IV and DNA-protein kinase (DNA-PK), playing crucial roles in the nonhomologous end joining (NHEJ) pathway, which is the predominant pathway for DNA double-strand break repair (DNA-DSB-repair) in mammalian cells. DNA-PK(+/+) as well as DNA-ligase IV (+/+) cancer cells were apoptosis resistant and deficient in activation of caspase-3, caspase-9, and caspase-8 and in cleavage of poly(ADP-ribose) polymerase after doxorubicin treatment. Inhibition of NHEJ by knocking out DNA-PK or DNA-ligase IV restored caspases activation and apoptosis sensitivity after doxorubicin treatment. In addition, inhibition of caspases activation prevented doxorubicin-induced apoptosis but could not prevent doxorubicin-induced DNA damage, indicating that induction of DNA damage is independent of caspases activation. However, caspases activation depends on induction of DNA damage left unrepaired by NHEJ-DNA-DSB-repair. We conclude that DNA damage left unrepaired by DNA-ligase IV or DNA-PK might be the initiator for caspases activation by doxorubicin in cancer cells. Failure in caspases activation using doxorubicin depends on loss of DNA damage and is due to higher rates of NHEJ-DNA-DBS-repair.

  16. CDP-choline reduces pro-caspase and cleaved caspase-3 expression, nuclear DNA fragmentation, and specific PARP-cleaved products of caspase activation following middle cerebral artery occlusion in the rat.

    PubMed

    Krupinski, J; Ferrer, I; Barrachina, M; Secades, J J; Mercadal, J; Lozano, R

    2002-05-01

    Citicoline has been demonstrated to be beneficial in several models of cerebral ischaemia. We tested the hypothesis that citicoline may provide apoptotic pathways following focal cerebral ischaemia. Focal cerebral ischaemia was produced by distal, permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. The animals were randomised into four groups: (B+A) Citicoline 500 mg/kg IP 24 and 1 h before MCAO, and 23 h after MCAO; (A) citicoline 500 mg/kg IP, within 30 min after MCAO, and 23 h after MCAO; (C) vehicle IP; and (D) sham-operated. The animals were sacrificed at 12 h (n=8 per group) and 24 h (n=8 per group) after MCAO. Immunohistochemistry was performed on free-floating tissue sections with goat polyclonal antibodies to procaspase-1, -2, -3, -6 and -8, and in paraffin-embedded sections processed for cleaved caspase-3 (17 kDa) immunohistochemistry. Finally, some sections were stained with the method of in situ end-labelling of nuclear DNA fragmentation. For gel electrophoresis and Western blotting, antibodies to poly (ADP-ribose) polymerase (PARP) products of 89 kDa were used to reveal specific cleavage substrates of caspases. MCAO induced the expression of all procaspases and the expression of PARP products of 89 kDa, as well as cells with nuclear DNA fragmentation, at 12 and 24 h, in the infarcted core and penumbra. Citicoline reduced the expression of all procaspases at 12 and 24 h after MCAO, as well as the expression of cleaved caspase-3 in cells in the penumbra area. This was accompanied by a reduction in the number of cells bearing nuclear DNA fragments. The expression of caspase-cleaved products of PARP (PARP 89 kDa) was reduced in citicoline-treated ischaemic rats. These results show that citicoline inhibits the expression of proteins involved in apoptosis following MCAO.

  17. Apoptotic and nonapoptotic function of caspase 7 in spermatogenesis.

    PubMed

    Lei, Bin; Zhou, Xuming; Lv, Daojun; Wan, Bo; Wu, Huayan; Zhong, Liren; Shu, Fangpeng; Mao, Xiangming

    2017-01-01

    Recent studies have reported that caspase 7 has an apoptotic and nonapoptotic function. However, the relationship between caspase 7 and spermatogenesis remains unknown. This study aimed to investigate the possible function of caspase 7 during normal and abnormal spermatogenesis. The cleaved form of caspase 7 was detected in testis tissues at different postpartum times (5-14 weeks) by qRT-PCR, Western blot and immunohistochemistry (IHC). Then, the mice models of spermatogenic dysfunction were obtained by busulfan (30 mg kg-1 to further evaluate the potential function and mechanism of caspase 7. qRT-PCR and Western blot results showed that caspase 7 expression was gradually elevated from 5 to 14 weeks, which was not connected with apoptosis. IHC results revealed that caspase 7 was mainly located in spermatogenic cells and Leydig cells. In addition, spermatogenic dysfunction induced by busulfan gradually enhanced the apoptosis and elevated the expression of caspase 3, caspase 6, and caspase 9, but decreased the expression of caspase 7 in spermatogenic cells. However, when spermatogenic cells were mostly disappeared at the fourth week after busulfan treatment, caspase 7 expression in Leydig cells was significantly increased and positively correlated with the expression of caspase 3, caspase 6, and caspase 9. Therefore, these results indicate that caspase 7 has a nonapoptic function that participates in normal spermatogenesis, but also displays apoptotic function in spermatogenic dysfunction.

  18. TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

    PubMed Central

    Fritsch, Jürgen; Fickers, Ricarda; Klawitter, Jan; Särchen, Vinzenz; Zingler, Philipp; Adam, Dieter; Janssen, Ottmar; Krause, Eberhard; Schütze, Stefan

    2016-01-01

    During apoptosis induction by TNF, the extrinsic and intrinsic apoptosis pathways converge at the lysosomal-mitochondrial interface. Earlier studies showed that the lysosomal aspartic protease Cathepsin D (CtsD) cleaves Bid to tBid, resulting in the amplification of the initial apoptotic cascade via mitochondrial outer membrane permeabilization (MOMP). The goal of this study was to identify further targets for CtsD that might be involved in activation upon death receptor ligation. Using a proteomics screen, we identified the heat shock protein 90 (HSP90) to be cleaved by CtsD after stimulation of U937 or other cell lines with TNF, FasL and TRAIL. HSP90 cleavage corresponded to apoptosis sensitivity of the cell lines to the different stimuli. After mutation of the cleavage site, HSP90 partially prevented apoptosis induction in U937 and Jurkat cells. Overexpression of the cleavage fragments in U937 and Jurkat cells showed no effect on apoptosis, excluding a direct pro-apoptotic function of these fragments. Pharmacological inhibition of HSP90 with 17AAG boosted ligand mediated apoptosis by enhancing Bid cleavage and caspase-9 activation. Together, we demonstrated that HSP90 plays an anti-apoptotic role in death receptor signalling and that CtsD-mediated cleavage of HSP90 sensitizes cells for apoptosis. These findings identify HSP90 as a potential target for cancer therapy in combination with death ligands (e.g. TNF or TRAIL). PMID:27716614

  19. The equine arteritis virus induces apoptosis via caspase-8 and mitochondria-dependent caspase-9 activation.

    PubMed

    St-Louis, Marie-Claude; Archambault, Denis

    2007-10-10

    We have previously showed that equine arteritis virus (EAV), an arterivirus, induces apoptosis in vitro. To determine the caspase activation pathways involved in EAV-induced apoptosis, target cells were treated with peptide inhibitors of apoptosis Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8-specific inhibitor) or Z-LEHD-FMK (caspase-9-specific inhibitor) 4 h prior to infection with the EAV T1329 Canadian isolate. Significant inhibition of apoptosis was obtained with all peptide inhibitors used. Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given the activation of Bid and the translocation of cytochrome c within the cytoplasm, the overall results indicate that EAV induces apoptosis initiated by caspase-8 activation and subsequent mitochondria-dependent caspase-9 activation.

  20. Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death

    PubMed Central

    Thurston, Teresa L. M.; Matthews, Sophie A.; Jennings, Elliott; Alix, Eric; Shao, Feng; Shenoy, Avinash R.; Birrell, Mark A.; Holden, David W.

    2016-01-01

    Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria. PMID:27808091

  1. Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells.

    PubMed

    Häussermann, S; Kittstein, W; Rincke, G; Johannes, F J; Marks, F; Gschwendt, M

    1999-12-03

    Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

  2. Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

    SciTech Connect

    Jun, Do Youn; Kim, Jun Seok; Park, Hae Sun; Song, Woo Sun; Bae, Young Seuk; Kim, Young Ho . E-mail: ykim@knu.ac.kr

    2007-07-15

    To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 {mu}M) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins.

  3. Caspase-dependent and -independent suppression of apoptosis by monoHER in Doxorubicin treated cells

    PubMed Central

    Bruynzeel, A M E; Abou El Hassan, M A; Torun, E; Bast, A; van der Vijgh, W J F; Kruyt, F A E

    2007-01-01

    Doxorubicin (DOX) is an antitumour agent for different types of cancer, but the dose-related cardiotoxicity limits its clinical use. To prevent this side effect we have developed the flavonoid monohydroxyethylrutoside (monoHER), a promising protective agent, which did not interfere with the antitumour activity of DOX. To obtain more insight in the mechanism underlying the selective protective effects of monoHER, we investigated whether monoHER (1 mM) affects DOX-induced apoptosis in neonatal rat cardiac myocytes (NeRCaMs), human endothelial cells (HUVECs) and the ovarian cancer cell lines A2780 and OVCAR-3. DOX-induced cell death was effectively reduced by monoHER in heart, endothelial and A2780 cells. OVCAR-3 cells were highly resistant to DOX-induced apoptosis. Experiments with the caspase-inhibitor zVAD-fmk showed that DOX-induced apoptosis was caspase-dependent in HUVECs and A2780 cells, whereas caspase-independent mechanisms seem to be important in NeRCaMs. MonoHER suppressed DOX-dependent activation of the mitochondrial apoptotic pathway in normal and A2780 cells as illustrated by p53 accumulation and activation of caspase-9 and -3 cleavage. Thus, monoHER acts by suppressing the activation of molecular mechanisms that mediate either caspase-dependent or -independent cell death. In light of the current work and our previous studies, the use of clinically achievable concentrations of monoHER has no influence on the antitumour activity of DOX whereas higher concentrations as used in the present study could influence the antitumour activity of DOX. PMID:17285121

  4. Specificity of hammerhead ribozyme cleavage.

    PubMed Central

    Hertel, K J; Herschlag, D; Uhlenbeck, O C

    1996-01-01

    To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

  5. Identification of novel mammalian caspases reveals an important role of gene loss in shaping the human caspase repertoire.

    PubMed

    Eckhart, Leopold; Ballaun, Claudia; Hermann, Marcela; VandeBerg, John L; Sipos, Wolfgang; Uthman, Aumaid; Fischer, Heinz; Tschachler, Erwin

    2008-05-01

    Proteases of the caspase family play central roles in apoptosis and inflammation. Recently, we have described a new gene encoding caspase-15 that has been inactivated independently in different mammalian lineages. To determine the dynamics of gene duplication and loss in the entire caspase gene family, we performed a comprehensive evolutionary analysis of mammalian caspases. By comparative genomics and reverse transcriptase-polymerase chain reaction analyses, we identified 3 novel mammalian caspase genes, which we tentatively named caspases-16 through -18. Caspase-16, which is most similar in sequence to caspase-14, has been conserved in marsupials and placental mammals, including humans. Caspase-17, which is most similar to caspase-3, has been conserved among fish, frog, chicken, lizard, and the platypus but is absent from marsupials and placental mammals. Caspase-18, which is most similar to caspase-8, has been conserved among chicken, platypus, and opossum but is absent from placental mammals. These gene distribution patterns suggest that, in the evolutionary lineage leading to humans, caspase-17 was lost after the split of protherian and therian mammals and caspase-18 was lost after the split of marsupials and placental mammals. In the canine genome, the number of caspases has been reduced by the fusion of the neighboring genes caspases-1 and -4, resulting in a single coding region. Further lineage-specific gene inactivations were found for caspase-10 in murine rodents and caspase-12 in humans, rabbit, and cow. Lineage-specific gene duplications were found for caspases-1, -3, and -12 in opossum and caspase-4 in primates. Other caspases were generally conserved in all mammalian species investigated. Using the positions of introns as stable characters during recent vertebrate evolution, we define 3 phylogenetic clades of caspase genes: caspases-1/-2/-4/-5/-9/-12/-14/-15/-16 (clade I), caspases-3/-6/-7/-17 (clade II), and caspases-8/-10/-18/CFLAR (clade III). We

  6. Epigenetic regulation of nitric oxide synthase 2, inducible (Nos2) by NLRC4 inflammasomes involves PARP1 cleavage.

    PubMed

    Buzzo, Carina de Lima; Medina, Tiago; Branco, Laura M; Lage, Silvia L; Ferreira, Luís Carlos de Souza; Amarante-Mendes, Gustavo P; Hottiger, Michael O; De Carvalho, Daniel D; Bortoluci, Karina R

    2017-02-02

    Nitric oxide synthase 2, inducible (Nos2) expression is necessary for the microbicidal activity of macrophages. However, NOS2 over-activation causes multiple inflammatory disorders, suggesting a tight gene regulation is necessary. Using cytosolic flagellin as a model for inflammasome-dependent NOS2 activation, we discovered a surprising new role for NLRC4/caspase-1 axis in regulating chromatin accessibility of the Nos2 promoter. We found that activation of two independent mechanisms is necessary for NOS2 expression by cytosolic flagellin: caspase-1 and NF-κB activation. NF-κB activation was necessary, but not sufficient, for NOS2 expression. Conversely, caspase-1 was necessary for NOS2 expression, but dispensable for NF-κB activation, indicating that this protease acts downstream NF-κB activation. We demonstrated that epigenetic regulation of Nos2 by caspase-1 involves cleavage of the chromatin regulator PARP1 (also known as ARTD1) and chromatin accessibility of the NF-κB binding sites located at the Nos2 promoter. Remarkably, caspase-1-mediated Nos2 transcription and NO production contribute to the resistance of macrophages to Salmonella typhimurium infection. Our results uncover the molecular mechanism behind the constricted regulation of Nos2 expression and open new therapeutic opportunities based on epigenetic activities of caspase-1 against infectious and inflammatory diseases.

  7. Epigenetic regulation of nitric oxide synthase 2, inducible (Nos2) by NLRC4 inflammasomes involves PARP1 cleavage

    PubMed Central

    Buzzo, Carina de Lima; Medina, Tiago; Branco, Laura M.; Lage, Silvia L.; Ferreira, Luís Carlos de Souza; Amarante-Mendes, Gustavo P.; Hottiger, Michael O.; De Carvalho, Daniel D.; Bortoluci, Karina R.

    2017-01-01

    Nitric oxide synthase 2, inducible (Nos2) expression is necessary for the microbicidal activity of macrophages. However, NOS2 over-activation causes multiple inflammatory disorders, suggesting a tight gene regulation is necessary. Using cytosolic flagellin as a model for inflammasome-dependent NOS2 activation, we discovered a surprising new role for NLRC4/caspase-1 axis in regulating chromatin accessibility of the Nos2 promoter. We found that activation of two independent mechanisms is necessary for NOS2 expression by cytosolic flagellin: caspase-1 and NF-κB activation. NF-κB activation was necessary, but not sufficient, for NOS2 expression. Conversely, caspase-1 was necessary for NOS2 expression, but dispensable for NF-κB activation, indicating that this protease acts downstream NF-κB activation. We demonstrated that epigenetic regulation of Nos2 by caspase-1 involves cleavage of the chromatin regulator PARP1 (also known as ARTD1) and chromatin accessibility of the NF-κB binding sites located at the Nos2 promoter. Remarkably, caspase-1-mediated Nos2 transcription and NO production contribute to the resistance of macrophages to Salmonella typhimurium infection. Our results uncover the molecular mechanism behind the constricted regulation of Nos2 expression and open new therapeutic opportunities based on epigenetic activities of caspase-1 against infectious and inflammatory diseases. PMID:28150715

  8. Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

    NASA Astrophysics Data System (ADS)

    Lee, Hyeran; Akers, Walter J.; Cheney, Philip P.; Edwards, W. Barry; Liang, Kexian; Culver, Joseph P.; Achilefu, Samuel

    2009-07-01

    Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with 64Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters kcat and KM of 0.55+/-0.01 s-1 and 1.12+/-0.06 μM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled 64Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

  9. Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radiolabeled multimodality molecular probe

    PubMed Central

    Lee, Hyeran; Akers, Walter J.; Cheney, Philip P.; Edwards, W. Barry; Liang, Kexian; Culver, Joseph P.; Achilefu, Samuel

    2010-01-01

    Based on the capability of modulating fluorescence intensity by specific molecular events, we have developed a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of DOTA for chelating a radionuclide, a near infrared fluorescent dye, and an efficient quencher dye. The two dyes were separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 was radiolabeled with 64Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence was quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay showed that LS498 was readily cleaved by caspase-3, with excellent enzyme kinetic parameters kcat and KM of 0.55 ± 0.01 s−1 and 1.12 ± 0.06 μM, respectively. In mice, the initial fluorescence of LS498 was 10-fold less than control. Using radiolabeled 64Cu-LS498 in a controlled and localized in vivo model of caspase-3 activation, a time-dependent 5-fold NIR fluorescence enhancement was observed but radioactivity was the same in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes. PMID:19725712

  10. PARP and RIP 1 are required for autophagy induced by 11'-deoxyverticillin A, which precedes caspase-dependent apoptosis.

    PubMed

    Zhang, Nan; Chen, Yali; Jiang, Ruixuan; Li, Erwei; Chen, Xiuling; Xi, Zhijun; Guo, Yinglu; Liu, Xingzhong; Zhou, Yuguang; Che, Yongsheng; Jiang, Xuejun

    2011-06-01

    The epipolythiodioxopiperazines (ETPs) are fungal secondary metabolites proven to trigger both apoptotic and necrotic cell death of tumor cells. However, the underlying mechanism of their regulatory role in macroautophagy and the interplay between autophagy and apoptosis initiated by the ETPs, remain unexplored. In the current work, we found that 11'-deoxyverticillin A (C42), a member of the ETPs, induces autophagosome formation, accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II ) and degradation of sequestosome 1 (SQSTM1/p62). In addition, the LC3-II accrual and p62 degradation occur prior to caspase activation and coincide with PARP activation. Inhibition of autophagy by either chemical inhibitors or by RNA interference single knockdown of essential autophagic genes partially reduces the cell death and the cleavage of both caspase 3 and PARP. Necrostatin-1, a specific inhibitor of necroptosis, inhibits both the augmentation of LC3-II and the cleavage of caspase 3, which was confirmed by depletion of receptor-interacting protein 1 (RIP-1), a crucial necrostatin-1-targeted adaptor kinase mediating cell death and survival. Moreover, inhibition of PARP by either chemical inhibitors or RNA interference provides obvious protection for cell viability and suppresses the LC3-II accretion caused by C42 treatment. Interestingly, double silencing of LC3 and p62 completely suppressed PARP cleavage and concurrently and maximally augmented the PAR formation induced by C42. Collectively, we have demonstrated that C42 enhances the cellular autophagic process, which requires both PARP and RIP-1 participation, preceding and possibly augmenting, the caspase-dependent apoptotic cell death.

  11. Caspase-resistant vimentin suppresses apoptosis after photodynamic treatment with a silicon phthalocyanine in Jurkat cells.

    PubMed

    Belichenko, I; Morishima, N; Separovic, D

    2001-06-01

    Oxidative stress, such as photodynamic therapy, is an apoptosis inducer. Apoptosis, as well as photosensitization, have been associated with disruption of the cytoskeletal network. The purpose of the present study was to assess the role of vimentin, a major cytoskeletal protein, in apoptosis after photodynamic treatment (PDT) with the silicon phthalocyanine Pc 4 in human Jurkat T cells. Here we show for the first time that photosensitization with Pc 4 initiates vimentin cleavage and that this event precedes poly(ADP-ribose) polymerase (PARP) degradation. Similar findings were obtained in the presence of C2-ceramide, an inducer of oxidative stress and apoptosis. In the presence of benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, a pan-caspase inhibitor, Pc 4-PDT-induced vimentin and PARP cleavage were abolished. In Jurkat cells transfected with a caspase-resistant vimentin apoptosis was partly suppressed and delayed post-Pc 4-PDT. We suggest that the full-length vimentin confers resistance to nuclear apoptosis after PDT with Pc 4.

  12. Internalized Cryptococcus neoformans Activates the Canonical Caspase-1 and the Noncanonical Caspase-8 Inflammasomes.

    PubMed

    Chen, Mingkuan; Xing, Yue; Lu, Ailing; Fang, Wei; Sun, Bing; Chen, Changbin; Liao, Wanqing; Meng, Guangxun

    2015-11-15

    Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcosis in immunocompromised patients as well as immunocompetent individuals. Host cell surface receptors that recognize C. neoformans have been widely studied. However, intracellular sensing of this pathogen is still poorly understood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome. In the current study, it was found that opsonization-mediated internalization of encapsulated C. neoformans also activated the canonical NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC)-caspase-1 inflammasome. In addition, the internalized C. neoformans activated the noncanonical NLRP3-ASC-caspase-8 inflammasome as well, which resulted in robust IL-1β secretion and cell death from caspase-1-deficient primary dendritic cells. Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further mechanistic studies showed that both phagolysosome membrane permeabilization and potassium efflux were responsible for C. neoformans-induced activation of either the canonical NLRP3-ASC-caspase-1 inflammasome or the noncanonical NLRP3-ASC-caspase-8 inflammasome. Moreover, challenge with zymosan also led to the activation of the noncanonical NLRP3-ASC-caspase-8 inflammasome in cells absent for caspase-1. Collectively, these findings uncover a number of novel signaling pathways for the innate immune response of host cells to C. neoformans infection and suggest that manipulating NLRP3 signaling may help to control fungal challenge.

  13. Procaspase-9 induces its cleavage by transnitrosylating XIAP via the Thioredoxin system during cerebral ischemia-reperfusion in rats

    PubMed Central

    Zhang, Dengyue; Zhao, Ningjun; Ma, Bin; Wang, Yan; Zhang, Gongliang; Yan, Xianliang; Hu, Shuqun; Xu, Tie

    2016-01-01

    Transnitrosylation is an important mechanism by which nitric oxide (NO) modulates cell signaling pathways. For instance, SNO-caspase-3 can transnitrosylate the X-linked inhibitor of apoptosis (XIAP) to enhance apoptosis. XIAP is a potent antagonist of caspase apoptotic activity. Decrease in XIAP activity via nitrosylation results in SNO-XIAP-mediated caspase activation. Considering the functional liaison of procaspase-9 and XIAP, we hypothesized that procaspase-9 nitrosylates XIAP directly. Our data confirmed that cerebral ischemia-reperfusion induced XIAP nitrosylation, procaspase-9 denitrosylation and cleavage. Interestingly, the time courses of the nitrosylation of procaspase-9 and XIAP were negatively correlated, which was more prominent after cerebral ischemia-reperfusion, suggesting a direct interaction. The nitrosylation of XIAP, as well as the denitrosylation and cleavage of procaspase-9, were inhibited by DNCB, TrxR1 AS-ODNs, or TAT-AVPY treatment. Meanwhile, DNCB, TrxR1 AS-ODNs, or TAT-AVPY also inhibited the decrease in hippocampal CA1 neurons induced by ischemia-reperfusion in rats. The denitrosylation and cleavage of procaspase-9 induced by OGD/reoxygenation in SH-SY5Y cells were inhibited when cells were co-transfected with wild-type procaspase-9 and XIAP mutant (C449G). These data suggest that cerebral ischemia-reperfusion induces a transnitrosylation from procaspase-9 to XIAP via the Trx system to consequently cause apoptosis. Additionally, Cys325 is a critical S-nitrosylation site of procaspase-9. PMID:27052476

  14. Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors

    SciTech Connect

    Wang, Zhigang; Watt, William; Brooks, Nathan A.; Harris, Melissa S.; Urban, Jan; Boatman, Douglas; McMillan, Michael; Kahn, Michael; Heinrikson, Robert L.; Finzel, Barry C.; Wittwer, Arthur J.; Blinn, James; Kamtekar, Satwik; Tomasselli, Alfredo G.

    2010-09-17

    Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel {beta}-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.

  15. A role for caspase-8 and c-FLIPL in proliferation and cell-cycle progression of primary hepatocytes.

    PubMed

    Gilot, David; Serandour, Anne-Laure; Ilyin, Guennady P; Lagadic-Gossmann, Dominique; Loyer, Pascal; Corlu, Anne; Coutant, Alexandre; Baffet, Georges; Peter, Marcus E; Fardel, Olivier; Guguen-Guillouzo, Christiane

    2005-12-01

    Growth factors are known to favor both proliferation and survival of hepatocytes. In the present study, we investigated if c-FLIP(L) (cellular FLICE-inhibitory protein, long isoform) could be involved in epidermal growth factor (EGF)-stimulated proliferation of rat hepatocytes since c-FLIP(L) regulates both cell proliferation and procaspase-8 maturation. Treatment with MEK inhibitors prevented induction of c-FLIP(L) by EGF along with total inhibition of DNA replication. However, EGF failed to inhibit processing of procaspase-8 in the presence of EGF suggesting that c-FLIP(L) does not play its canonical anti-apoptotic role in this model. Downregulation of c-FLIP expression using siRNA oligonucleotides strongly reduced DNA replication but did not result in enhanced apoptosis. Moreover, intermediate cleavage products of c-FLIP(L) and caspase-8 were found in EGF-treated hepatocytes in the absence of caspase-3 maturation and cell death. To determine whether the Fas/FADD/caspase-8/c-FLIP(L) complex was required for this activity, Fas, procaspase-8 and Fas-associated death domain protein (FADD) expression or function was inhibited using siRNA or constructs encoding dominant negative mutant proteins. Inhibition of any of these components of the Fas/FADD/caspase-8 pathway decreased DNA replication suggesting a function of these proteins in cell-cycle arrest. Similar results were obtained when the IETD-like caspase activity detectable in EGF-treated hepatocytes was inhibited by the pan-caspase inhibitor, z-ASP. Finally, we demonstrated co-immunoprecipitation between EGFR and Fas within 15 min following EGF stimulation. In conclusion, our results indicate that the Fas/FADD/c-FLIP(L)/caspase-8 pathway positively controls the G(1)/S transition in EGF-stimulated hepatocytes. Our data provide new insights into the mechanisms by which apoptotic proteins participate to mitogenic signals during the G(1) phase.

  16. Silencing of Pokemon Enhances Caspase-Dependent Apoptosis via Fas- and Mitochondria-Mediated Pathways in Hepatocellular Carcinoma Cells

    PubMed Central

    Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy. PMID:23874836

  17. Signal transduction activated by the cancer chemopreventive isothiocyanates: cleavage of BID protein, tyrosine phosphorylation and activation of JNK

    PubMed Central

    Xu, K; Thornalley, P J

    2001-01-01

    Phenethyl isothiocyanate and allyl isothiocyanate induce apoptosis of human leukaemia HL60 cells in vitro. Apoptosis was associated with cleavage of p22 BID protein to p15, p13 and p11 fragments and activation of JNK and tyrosine phosphorylation (18 kDa and 45 kDa proteins). All these effects and apoptosis were prevented by exogenous glutathione (15 mM). Protein tyrosine phosphatase activity was unchanged. The general caspase inhibitor Z-VAD-fmk prevented apoptosis but not JNK activation – excluding a role for caspases in JNK activation, whereas curcumin prevented JNK activation but only delayed apoptosis. This suggests that in isothiocyanate-induced apoptosis, the caspase pathway has an essential role, the JNK pathway a supporting role, and inhibition of protein tyrosine phosphatases is not involved. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11237388

  18. Krebs Cycle Moonlights in Caspase Regulation.

    PubMed

    Minis, Adi; Steller, Hermann

    2016-04-04

    In this issue of Developmental Cell, Aram et al. (2016) identify a mechanism that uses a Krebs cycle protein to control local activation of a ubiquitin ligase complex at the mitochondrial outer membrane for temporally and spatially restricted caspase activation during Drosophila sperm differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Caspases inhibition decreases neurological sequelae in meningitis.

    PubMed

    Irazuzta, Jose; Pretzlaff, Robert K; Zingarelli, Basilia

    2008-05-01

    To evaluate the effects of sustained caspase inhibition during the acute phase of meningitis-induced brain injury. Changes in neurobehavioral performance were the primary outcome variables. Randomized prospective animal study. University research laboratory. Male Wistar rats. Animals underwent a basilar cistern inoculation of group B Streptococci to induce meningitis. Sixteen hours later animals were randomized to receive Bocaspartyl (OMe)-fluoromethyketone (BAF) for 4 days or placebo in addition to antibiotic therapy. The assessment of neurobehavioral performance was started 7 days after initiation of treatment and continued for the following 3 wks. A subgroup underwent early kill, at 5 days, to evaluate caspase 3 activity in brain tissue. There was a group of Sham instrumented animals. BAF decreased caspase 3 activation in meningitic animals. There were no significant motor deficit differences between the infected groups. Cognitive performance was significantly improved in the BAF group. These findings demonstrate that sustained systemic administration of BAF inhibits caspase 3 activation and decreases neurologic sequelae in a rat model of bacterial meningitis.

  20. Thalidomide inhibits activation of caspase-1.

    PubMed

    Keller, Martin; Sollberger, Gabriel; Beer, Hans-Dietmar

    2009-11-01

    Thalidomide is an efficient anti-inflammatory and anti-angiogenic drug, but its therapeutic use is problematic due to a strong teratogenic activity. Nevertheless, thalidomide was approved for the treatment of inflammatory skin diseases and certain types of cancer, and it is extensively tested for several other indications. Recently, we demonstrated that active caspase-1, whose activation is dependent on inflammasome complexes, is required for unconventional protein secretion of proinflammatory cytokines such as IL-1 and of the proangiogenic fibroblast growth factor 2. In this study, we show that pharmacological doses of thalidomide strongly reduced the secretion of both proteins. Thalidomide-treated cells also released less of other leaderless proteins, which require caspase-1 activity for their secretion. In line with these findings, the drug inhibited activation and activity of caspase-1 in cultured cells but not in vitro. The latter finding suggests that the pharmacological activity is exerted by a metabolite of the drug. The anti-inflammatory activity of thalidomide was also mediated via caspase-1 in mice. These findings represent a novel mechanism by which thalidomide exerts its pharmacological activity and suggest that inhibition of the activity of IL-1 might represent a novel strategy to substitute thalidomide.

  1. Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein.

    PubMed

    Nervi, C; Ferrara, F F; Fanelli, M; Rippo, M R; Tomassini, B; Ferrucci, P F; Ruthardt, M; Gelmetti, V; Gambacorti-Passerini, C; Diverio, D; Grignani, F; Pelicci, P G; Testi, R

    1998-10-01

    All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARalpha product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARalpha and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARalpha (U937/PR9) and in blasts from APL patients, the PML/RARalpha fusion protein is cleaved by a caspase 3-like activity induced by RA treatment. In fact, a caspase 3-like activity is detectable in PML/RARalpha expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARalpha deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the alpha-helix region of the PML component of the fusion protein. The extent of PML/RARalpha cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARalpha is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARalpha.

  2. Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro

    PubMed Central

    Jiang, Shan; Xie, Qing; Zhang, Wei; Zhou, Xia-Qiu; Jin, You-Xin

    2005-01-01

    AIM: To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis. METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neorpBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with [α-32P]UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37°C in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg2+). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE (containing 8 mol/L urea). RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37°C, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%. CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis. PMID:15996037

  3. Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6.

    PubMed

    Karvinen, Jarkko; Elomaa, Annika; Mäkinen, Maija Liisa; Hakala, Harri; Mukkala, Veli Matti; Peuralahti, Jari; Hurskainen, Pertti; Hovinen, Jari; Hemmilä, Ilkka

    2004-02-15

    Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.

  4. CASPASE CONTROL: PROTAGONISTS OF CANCER CELL APOPTOSIS

    PubMed Central

    Fiandalo, M.V.; Kyprianou, N.

    2013-01-01

    Emergence of castration-resistant metastatic prostate cancer is due to activation of survival pathways, including apoptosis suppression and anoikis resistance, and increased neovascularization. Thus targeting of apoptotic players is of critical significance in prostate cancer therapy since loss of apoptosis and resistance to anoikis are critical in aberrant malignant growth, metastasis and conferring therapeutic failure. The majority of therapeutic agents act through intrinsic mitochondrial, extrinsic death receptor pathways or endoplasmic reticulum stress pathways to induce apoptosis. Current therapeutic strategies target restoring regulatory molecules that govern the pro-survival pathways such as PTEN which regulates AKT activity. Other strategies focus on reactivating the apoptotic pathways either by down-regulating anti-apoptotic players such as BCL-2 or by up-regulating pro-apoptotic protein families, most notably, the caspases. Caspases are a family of cystine proteases which serve critical roles in apoptotic and inflammatory signaling pathways. During tumorigenesis, significant loss or inactivation of lead members in the caspase family leads to impairing apoptosis induction, causing a dramatic imbalance in the growth dynamics, ultimately resulting in aberrant growth of human cancers. Recent exploitation of apoptosis pathways towards re-instating apoptosis induction via caspase re-activation has provided new molecular platforms for the development of therapeutic strategies effective against advanced prostate cancer as well as other solid tumors. This review will discuss the current cellular landscape featuring the caspase family in tumor cells and their activation via pharmacologic intervention towards optimized anti-cancer therapeutic modalities. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”. PMID:23070001

  5. The Tick Salivary Protein Sialostatin L2 Inhibits Caspase-1-Mediated Inflammation during Anaplasma phagocytophilum Infection

    PubMed Central

    Chen, Gang; Wang, Xiaowei; Severo, Maiara S.; Sakhon, Olivia S.; Sohail, Mohammad; Brown, Lindsey J.; Sircar, Mayukh; Snyder, Greg A.; Sundberg, Eric J.; Ulland, Tyler K.; Olivier, Alicia K.; Andersen, John F.; Zhou, Yi; Shi, Guo-Ping; Sutterwala, Fayyaz S.; Kotsyfakis, Michail

    2014-01-01

    Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1β (IL-1β) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor κB or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology. PMID:24686067

  6. Serial killers: ordering caspase activation events in apoptosis.

    PubMed

    Slee, E A; Adrain, C; Martin, S J

    1999-11-01

    Caspases participate in the molecular control of apoptosis in several guises; as triggers of the death machinery, as regulatory elements within it, and ultimately as a subset of the effector elements of the machinery itself. The mammalian caspase family is steadily growing and currently contains 14 members. At present, it is unclear whether all of these proteases participate in apoptosis. Thus, current research in this area is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of cell death. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation. As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling (upstream) and effector (downstream) roles based upon their prodomain lengths. In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme. Here, we discuss the apoptosis-associated caspase cascade and the hierarchy of caspase activation events within it.

  7. Microstructure and cleavage in lath martensitic steels

    NASA Astrophysics Data System (ADS)

    Morris, John W., Jr.; Kinney, Chris; Pytlewski, Ken; Adachi, Y.

    2013-02-01

    In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified ‘classic’ lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov-Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage.

  8. Microstructure and cleavage in lath martensitic steels.

    PubMed

    Morris, John W; Kinney, Chris; Pytlewski, Ken; Adachi, Y

    2013-02-01

    In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified 'classic' lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov-Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage.

  9. Kinetics of hairpin ribozyme cleavage in yeast.

    PubMed Central

    Donahue, C P; Fedor, M J

    1997-01-01

    Hairpin ribozymes catalyze a self-cleavage reaction that provides a simple model for quantitative analyses of intracellular mechanisms of RNA catalysis. Decay rates of chimeric mRNAs containing self-cleaving ribozymes give a direct measure of intracellular cleavage kinetics in yeast. Intracellular ribozyme-mediated cleavage occurs at similar rates and shows similar inhibition by ribozyme mutations as ribozyme-mediated reactions in vitro, but only when ribozymes are located in a favorable mRNA sequence context. The impact of cleavage on mRNA abundance is shown to depend directly on intrinsic mRNA stability. Surprisingly, cleavage products are no more labile than uncleaved mRNAs despite the loss of terminal cap structures or poly (A). PMID:9292496

  10. Necrosis- and apoptosis-related Met cleavages have divergent functional consequences

    PubMed Central

    Montagne, R; Berbon, M; Doublet, L; Debreuck, N; Baranzelli, A; Drobecq, H; Leroy, C; Delhem, N; Porte, H; Copin, M-C; Dansin, E; Furlan, A; Tulasne, D

    2015-01-01

    Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains (calcium-dependent proteases). These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged. PMID:25996296

  11. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3.

    PubMed

    Toonen, Lodewijk J A; Schmidt, Iris; Luijsterburg, Martijn S; van Attikum, Haico; van Roon-Mom, Willeke M C

    2016-10-12

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3.

  12. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3

    PubMed Central

    Toonen, Lodewijk J. A.; Schmidt, Iris; Luijsterburg, Martijn S.; van Attikum, Haico; van Roon-Mom, Willeke M. C.

    2016-01-01

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3. PMID:27731380

  13. Sam68 is cleaved by caspases under apoptotic cell death induced by ionizing radiation.

    PubMed

    Cho, Seong-Jun; Choi, Moo Hyun; Nam, Seon Young; Kim, Ji Young; Kim, Cha Soon; Pyo, Suhkneung; Yang, Kwang Hee

    2015-03-01

    The RNA-binding protein Sam68, a mitotic substrate of tyrosine kinases, has been reported to participate in the cell cycle, apoptosis, and signaling. In particular, overexpression of Sam68 protein is known to suppress cell growth and cell cycle progression in NIH3T3 cells. Although Sam68 is involved in many cellular activities, the function of Sam68, especially in response to apoptotic stimulation, is not well understood. In this study, we found that Sam68 protein is cleaved in immune cells undergoing apoptosis induced by γ-radiation. Moreover, we found that Sam68 cleavage was induced by apoptotic stimuli containing γ-radiation in a caspase-dependent manner. In particular, we showed that activated casepase-3, 7, 8 and 9 can directly cleave Sam68 protein through in vitro protease cleavage assay. Finally, we found that the knockdown of Sam68 attenuated γ-radiation-induced cell death and growth suppression. Conclusively, the cleavage of Sam68 is a new indicator for the cell damaging effects of ionizing radiation. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  14. Fenretinide Activates Caspases and Induces Apoptosis in Gliomas

    PubMed Central

    Puduvalli, Vinaykumar K.; Saito, Yoshiki; Xu, Ruishu; Kouraklis, Gregory P.; Levin, Victor A.; Kyritsis, Athanassios P.

    2014-01-01

    The synthetic retinoid fenretinide (N-[4-hydroxyphenyl] retinamide or 4HPR) has been shown to not only inhibit cell growth but also to induce apoptosis in a variety of malignant cell lines. It is being tested presently for its potential as a chemopreventive agent against several cancers. A related retinoid, 13-cis-retinoic acid (cRA), has been shown to have activity against gliomas in vitro as well as in a recent clinical study. The present study aimed at assessing the activity of fenretinide against glioma cells in vitro and comparing it with that of cRA at pharmacologically relevant doses. We hypothesized that the ability of fenretinide to induce apoptosis would make it more potent against gliomas than cRA. Four glioma cell lines (D54, U251, U87MG, and EFC-2) were treated with fenretinide (1–100 µM) and showed dose- and time-dependent induction of cell death. At pharmacologically relevant doses, fenretinide was more active against glioma cells than cRA because of its ability to induce apoptosis. Flow cytometric studies using D54 cells demonstrated no significant changes in the cell cycle distribution compared with untreated control, but a sub-G1 fraction consistent with apoptosis was detected. Terminal deoxynucleotidyl transferase-mediated nick end labeling assay indicated that the apoptotic fraction was cell cycle nonspecific. Fenretinide treatment resulted in cleavage of poly ADP-ribose polymerase, indicating an activation of the caspase 3. Immunofluorescence studies using the nuclear stain 4′,6-diamidine-2′-phenylindole dihydrochloride showed nuclear condensation and an apoptotic morphology. Hence, this study demonstrates that, at clinically relevant doses, fenretinide is a potent inducer of apoptosis in gliomas acting via the caspase pathway. We also show that at clinically achievable doses, fenretinide has more activity against gliomas than comparable doses of cRA. The favorable side effect profile seen in previous clinical studies and the in vitro

  15. Conformational similarity in the activation of caspase-3 and -7 revealed by the unliganded and inhibited structures of caspase-7

    SciTech Connect

    Agniswamy, Johnson; Fang, Bin; Weber, Irene T.

    2009-09-08

    Caspase-mediated apoptosis has important roles in normal cell differentiation and aging and in many diseases including cancer, neuromuscular disorders and neurodegenerative diseases. Therefore, modulation of caspase activity and conformational states is of therapeutic importance. We report crystal structures of a new unliganded conformation of caspase-7 and the inhibited caspase-7 with the tetrapeptide Ac-YVAD-Cho. Different conformational states and mechanisms for substrate recognition have been proposed based on unliganded structures of the redundant apoptotic executioner caspase-3 and -7. The current study shows that the executioner caspase-3 and -7 have similar conformations for the unliganded active site as well as the inhibitor-bound active site. The new unliganded caspase-7 structure exhibits the tyrosine flipping mechanism in which the Tyr230 has rotated to block entry to the S2 binding site similar to the active site conformation of unliganded caspase-3. The inhibited structure of caspase-7/YVAD shows that the P4 Tyr binds the S4 region specific to polar residues at the expense of a main chain hydrogen bond between the P4 amide and carbonyl oxygen of caspase-7 Gln 276, which is similar to the caspase-3 complex. This new knowledge of the structures and conformational states of unliganded and inhibited caspases will be important for the design of drugs to modulate caspase activity and apoptosis.

  16. Proteases for Cell Suicide: Functions and Regulation of Caspases

    PubMed Central

    Chang, Howard Y.; Yang, Xiaolu

    2000-01-01

    Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1β, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits. PMID:11104820

  17. Expression and prognostic significance of APAF-1, caspase-8 and caspase-9 in stage II/III colon carcinoma: caspase-8 and caspase-9 is associated with poor prognosis.

    PubMed

    Sträter, Jörn; Herter, Ines; Merkel, Gaby; Hinz, Ulf; Weitz, Jürgen; Möller, Peter

    2010-08-15

    Apoptosis protease activating factor-1 (APAF-1), caspase-8 and caspase-9 are important factors in the execution of death signals. To study their prognostic influence in colon carcinoma, expression of APAF-1, caspase-8 and caspase-9 was determined by immunohistochemistry in normal colon mucosa (n = 8) and R0-resected stage II/III colon carcinomas (n >or= 124) using a semiquantitative score. Staining results were correlated with disease-free survival by Kaplan-Meier estimates, and multivariate Cox analyses were performed. In normal colon, APAF-1 and caspase-8 are most strongly expressed in the luminal surface epithelium, whereas caspase-9 is expressed all along the crypt axis. In colon carcinomas, there is considerable variability in the expression of these proapoptotic factors, although complete loss of caspase-8 and caspase-9 is rare. APAF-1 expression did not correlate with disease-free survival. Instead, both expression of caspase-9 and high-level expression of caspase-8 in a majority of tumor cells were significantly associated with adverse prognosis (p = 0.004 and p = 0.029, respectively). The influence of caspase-8 expression was mainly seen in patients with stage III colon carcinoma (p = 0.011), whereas the prognostic influence of caspase-9 expression was significant in stage II cases (p = 0.037) and just failed to be significant in stage III tumors (p = 0.0581). After adjusting for confounding factors in a multivariate Cox analysis, the effect of caspase-9 in predicting disease-free survival was confirmed (p = 0.003). Our data suggest that, in colon carcinomas, expression of caspase-8 and caspase-9 is significantly associated with poor survival. Caspase-9 may be an independent prognosticator in colon carcinoma.

  18. Proteasomal regulation of caspase-8 in cancer cell apoptosis.

    PubMed

    Fiandalo, Michael V; Schwarze, Steven R; Kyprianou, Natasha

    2013-06-01

    Previous studies demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of caspase-8. The present study investigated the impact of proteasome inhibition on caspase-8 stability, ubiquitination, trafficking, and activation in cancer cells. Using caspase-8 deficient neuroblastoma (NB7) cells for reconstituting non-cleavable mutant forms of caspase-8, we demonstrated that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8, in response to proteasome inhibitor and GST-TRAIL. Moreover in the LNCaP human prostate cancer cells, caspase-8 polyubiquitination occurs after TRAIL stimulation and caspase-8 processing. Subcellular fractionation analysis revealed caspase-8 activity in both cytosol and plasma membrane fractions in both NB7 reconstituted caspase-8 cell lines, as well the LNCaP prostate cancer cells. The present results suggest that caspase-8 stabilization through proteasome inhibition leads to reactivation of the extrinsic pathway of apoptosis and identify E3 ligase mediating caspase-8 polyubiquitination, as a novel molecular target. Inhibition of this E3 ligase in combination with TRAIL towards restoring apoptosis signaling activation may have potential therapeutic significance in resistant tumors.

  19. Caspase-12 and the inflammatory response to Yersinia pestis.

    PubMed

    Ferwerda, Bart; McCall, Matthew B B; de Vries, Maaike C; Hopman, Joost; Maiga, Boubacar; Dolo, Amagana; Doumbo, Ogobara; Daou, Modibo; de Jong, Dirk; Joosten, Leo A B; Tissingh, Rudi A; Reubsaet, Frans A G; Sauerwein, Robert; van der Meer, Jos W M; van der Ven, André J A M; Netea, Mihai G

    2009-09-01

    Caspase-12 functions as an antiinflammatory enzyme inhibiting caspase-1 and the NOD2/RIP2 pathways. Due to increased susceptibility to sepsis in individuals with functional caspase-12, an early-stop mutation leading to the loss of caspase-12 has replaced the ancient genotype in Eurasia and a significant proportion of individuals from African populations. In African-Americans, it has been shown that caspase-12 inhibits the pro-inflammatory cytokine production. We assessed whether similar mechanisms are present in African individuals, and whether evolutionary pressures due to plague may have led to the present caspase-12 genotype population frequencies. No difference in cytokine induction through the caspase-1 and/or NOD2/RIP2 pathways was observed in two independent African populations, among individuals with either an intact or absent caspase-12. In addition, stimulations with Yersinia pestis and two other species of Yersinia were preformed to investigate whether caspase-12 modulates the inflammatory reaction induced by Yersinia. We found that caspase-12 did not modulate cytokine production induced by Yersinia spp. Our experiments demonstrate for the first time the involvement of the NOD2/RIP2 pathway for recognition of Yersinia. However, caspase-12 does not modulate innate host defense against Y. pestis and alternative explanations for the geographical distribution of caspase-12 should be sought.

  20. Proliferative versus Apoptotic Functions of Caspase-8 Hetero or Homo: The Caspase-8 Dimer Controls Cell Fate

    PubMed Central

    van Raam, Bram J.; Salvesen, Guy S.

    2014-01-01

    Caspase-8, the initiator of extrinsically-triggered apoptosis, also has important functions in cellular activation and differentiation downstream of a variety of cell surface receptors. It has become increasingly clear that the heterodimer of caspase-8 with the long isoform of cellular FLIP (FLIPL) fulfills these pro-survival functions of caspase-8. FLIPL, a catalytically defective caspase-8 paralog, can interact with caspase-8 to activate its catalytic function. The caspase-8/FLIPL heterodimer has a restricted substrate repertoire and does not induce apoptosis. In essence, caspase-8 heterodimerized with FLIPL prevents the receptor interacting kinases RIPK1 and -3 from executing the form of cell death known as necroptosis. This review discusses the latest insights in caspase-8 homo- vs. heterodimerization and the implication this has for cellular death or survival. PMID:21704196

  1. Cleavage plane determination in amphibian eggs.

    PubMed

    Sawai, T; Yomota, A

    1990-01-01

    In the present study using eggs of Cynops pyrrhogaster and Xenopus laevis, we examined (1) structural changes in the cytoplasm before the appearance of the cleavage furrow using a cytochemical method, (2) the time of cleavage plane determination depending on the mitotic apparatus (MA), by changing the shape of the eggs, and (3) the time of arrival of the "cleavage stimulus" at the cortex, by injecting colchicine solution or removing cytoplasm. Results were as follows: (1) In amphibian eggs the diastema was formed after development of the MA, appearing between the two asters after the MA had begun to degenerate. (2) The cleavage plane was preliminarily determined by the MA in the meta- to anaphase of karyokinesis. At this time, however, the egg cortex had not yet received the "cleavage stimulus" indispensable for furrow formation. (3) The egg cortex was really prepared to establish the furrow just after the edge of the diastema arrived at the cortex, when the MA had already degenerated. These results imply that the cleavage plane of the amphibian eggs is determined in two steps: the first, depending on the MA, is the determination of the direction of the growth of the diastema, and the second is the arrival of the "cleavage stimulus" at the cortex in association with the diastema.

  2. Geraniin-mediated apoptosis by cleavage of focal adhesion kinase through up-regulation of Fas ligand expression in human melanoma cells.

    PubMed

    Lee, Jang-Chang; Tsai, Chih-Yen; Kao, Jung-Yie; Kao, Ming-Ching; Tsai, Shih-Chang; Chang, Chih-Shiang; Huang, Li-Jiau; Kuo, Sheng-Chu; Lin, Jen-Kun; Way, Tzong-Der

    2008-06-01

    Geraniin, a form of tannin separated from geranium, causes cell death through induction of apoptosis; however, cell death characteristics for geraniin have not yet been elucidated. Here, we investigated the mechanism of geraniin-induced apoptosis in human melanoma cells and demonstrated that geraniin was able to induce cell apoptosis in a concentration- and time-dependent manner. We also examined the signaling pathway related to geraniin-induced apoptosis. To clarify the relationship between focal adhesion kinase (FAK) and geraniin-induced apoptosis, we treated human melanoma cells with geraniin and found that this resulted dose- and time-dependent degradation in FAK. However, FAK cleavage was significantly inhibited when cells were pretreated with a selective inhibitor of caspase-3 (Ac-Asp-Glu-Val-Asp-CHO). Here, we demonstrated for the first time that geraniin triggered cell death by caspase-3-mediated cleavage of FAK. There were two possible mechanisms for activating caspase-3, mitochondria-mediated and receptor-mediated apoptosis. To confirm the geraniin-relevant signaling pathway, using immunoblot analysis we found that geraniin-induced apoptosis was associated with the up-regulation of Fas ligand expression, the activation of caspase-8, the cleavage of Bid, and the induction of cytochrome c release from mitochondria to the cytosol. Treatment with geraniin caused induction of caspase-3 activity in a dose- and time-dependent manner followed by proteolytic cleavage of poly-(ADP-ribose) polymerase, and DNA fragmentation factor 45. The geraniin-induced apoptosis may provide a pivotal mechanism for its cancer-chemopreventive action.

  3. Dexamethasone decreases neurological sequelae and caspase activity.

    PubMed

    Irazuzta, Jose; Pretzlaff, Robert K; DeCourten-Myers, Gabrielle; Zemlan, Frank; Zingarelli, Basilia

    2005-01-01

    To evaluate the use of dexamethasone in a model of meningitis-induced brain injury. Changes in neurobehavioral performance were the primary outcome variables. Changes in caspase activation and markers of neuronal injury were the secondary outcome variables. Randomized, prospective animal study. University research laboratory. Male Wistar rats. Animals underwent a basilar cistern injection of either placebo or a suspension of Group B Streptococcus. Sixteen hours after inoculation, animals were randomized and received either dexamethasone or placebo in addition to antibiotics. Neurobehavioral performance and biological markers of brain injury were assessed at 3 days and 9 days after randomization. In a second experiment, caspase 1 and 3 were evaluated at 6 h, 24 h, and 72 h after dexamethasone administration. Neurobehavioral performance at 3 days and 9 days was significantly improved in the dexamethasone group. Serum C-tau and cerebral edema were decreased after 3 days of dexamethasone treatment. Dexamethasone decreased Caspase 3 activation in meningitic animals. These findings demonstrate that dexamethasone decreases acute brain injury in a rat model of bacterial meningitis as measured by preservation of neurobehavioral performance.

  4. Synergistic effect of fisetin combined with sorafenib in human cervical cancer HeLa cells through activation of death receptor-5 mediated caspase-8/caspase-3 and the mitochondria-dependent apoptotic pathway.

    PubMed

    Lin, Ming-Te; Lin, Chia-Liang; Lin, Tzu-Yu; Cheng, Chun-Wen; Yang, Shun-Fa; Lin, Chu-Liang; Wu, Chih-Chien; Hsieh, Yi-Hsien; Tsai, Jen-Pi

    2016-05-01

    Combining antitumor agents with bioactive compounds is a potential strategy for improving the effect of chemotherapy on cancer cells. The goal of this study was to elucidate the antitumor effect of the flavonoid, fisetin, combined with the multikinase inhibitor, sorafenib, against human cervical cancer cells in vitro and in vivo. The combination of fisetin and sorafenib synergistically induced apoptosis in HeLa cells, which is accompanied by a marked increase in loss of mitochondrial membrane potential. Apoptosis induction was achieved by caspase-3 and caspase-8 activation which increased the ratio of Bax/Bcl-2 and caused the subsequent cleavage of PARP level while disrupting the mitochondrial membrane potential in HeLa cells. Decreased Bax/Bcl-2 ratio level and mitochondrial membrane potential were also observed in siDR5-treated HeLa cells. In addition, in vivo studies revealed that the combined fisetin and sorafenib treatment was clearly superior to sorafenib treatment alone using a HeLa xenograft model. Our study showed that the combination of fisetin and sorafenib exerted better synergistic effects in vitro and in vivo than either agent used alone against human cervical cancer, and this synergism was based on apoptotic potential through a mitochondrial- and DR5-dependent caspase-8/caspase-3 signaling pathway. This combined fisetin and sorafenib treatment represents a novel therapeutic strategy for further clinical developments in advanced cervical cancer.

  5. Sinomenine induces apoptosis in RAW 264.7 cell-derived osteoclasts in vitro via caspase-3 activation

    PubMed Central

    He, Long-gang; Li, Xiang-lian; Zeng, Xiang-zhou; Duan, Heng; Wang, Song; Lei, Lin-sheng; Li, Xiao-juan; Liu, Shu-wen

    2014-01-01

    Aim: Sinomenine (SIN) is an alkaloid found in the roots and stems of Sinomenium acutum, which has been used to treat rheumatic arthritis in China and Japan. In this study we investigated the effects of SIN on osteoclast survival in vitro and the mechanisms of the actions. Methods: Mature osteoclasts were differentiated from murine monocyte/macrophage cell line RAW264.7 through incubation in the presence of receptor activator of NF-κB ligand (RANKL, 100 ng/mL) for 4 d. The cell viability was detected using the CCK-8 method. The survival and actin ring construction of the osteoclasts were scored using TRACP staining and phalloidin-FITC staining, respectively. The apoptosis of the osteoclasts was detected by DNA fragmentation and Hoechst 33258 staining, and the cell necrosis was indicated by LDH activity. The activation of caspase-3 in osteoclasts was measured using Western blotting and the caspase-3 activity colorimetric method. Results: SIN (0.25–2 mmol/L) inhibited the viability of mature osteoclasts in dose-dependent and time-dependent manners, but did not affect that of RAW264.7 cells. Consistently, SIN dose-dependently suppressed the survival of mature osteoclasts. The formation of actin ring, a marker associated with actively resorbing osteoclasts, was also impaired by the alkaloid. SIN (0.5 mmol/L) induced the apoptosis of mature osteoclasts, which was significantly attenuated in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. SIN increased the cleavage of caspase-3 in mature osteoclasts in dose-dependent and time-dependent manners. Furthermore, SIN dose-dependently enhanced caspase-3 activity, which was blocked in the presence of Ac-DEVD-CHO. Conclusion: Sinomenine inhibits osteoclast survival in vitro through caspase-3-mediated apoptosis, thus it is a potential agent for treating excessive bone resorption diseases. PMID:24362325

  6. Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3.

    PubMed

    Gao, Weijie; Xiao, Fenglian; Wang, Xiaoping; Chen, Tongsheng

    2013-10-01

    This report is designed to explore the roles of caspase-8, -9 and -3 in artemisinin (ARTE)-induced apoptosis in non-small cell lung cancer cells (A549 cells). ARTE induced reactive oxygen species (ROS)-mediated apoptosis in dose- and time-dependent fashion. Although ARTE treatment did not induce Bid cleavage and significant loss of mitochondrial membrane potential, it induced release of Smac and AIF but not cytochrome c from mitochondria, and silencing of Bak but not Bax significantly prevented ARTE-induced cytotoxicity. Moreover, ARTE treatment induced ROS-dependent activation of caspase-9, -8 and -3. Of the utmost importance, silencing or inhibiting any one of caspase-8, -9 and -3 almost completely prevented ARTE-induced activation of all the three caspases and remarkably abrogated the cytotoxicity of ARTE, suggesting that ARTE triggered an amplification activation loop among caspase-9, -8 and -3. Collectively, our data demonstrate that ARTE induces a ROS-mediated amplification activation loop among caspase-9, -8 and -3 to dominantly mediate the apoptosis of A549 cells.

  7. Highly sensitive detection of caspase-3 activities via a nonconjugated gold nanoparticle-quantum dot pair mediated by an inner-filter effect.

    PubMed

    Li, Jingwen; Li, Xinming; Shi, Xiujuan; He, Xuewen; Wei, Wei; Ma, Nan; Chen, Hong

    2013-10-09

    We describe here a simple fluorometric assay for the highly sensitive detection of caspase-3 activities on the basis of the inner-filter effect of gold nanoparticles (AuNPs) on CdTe quantum dots (QDs). The method takes advantage of the high molar absorptivity of the plasmon band of gold nanoparticles as well as the large absorption band shift from 520 to 680 nm upon nanoparticle aggregation. When labeled with a peptide possessing the caspase-3 cleavage sequence (DEVD), the monodispersed Au-Ps (peptide-modified AuNPs) exhibited a tendency to aggregate when exposed to caspase-3, which induced the absorption band transition from 520 to 680 nm and turned on the fluorescence of the CdTe QDs for caspase-3 sensing. Under optimum conditions, a high sensitivity towards caspase-3 was achieved with a detection limit as low as 18 pM, which was much lower than the corresponding assays based on absorbance or other approaches. Overall, we demonstrated a facile and sensitive approach for caspase-3 detection, and we expected that this method could be potentially generalized to design more fluorescent assays for sensing other bioactive entities.

  8. Bcl-2 and caspase-3 are major regulators in Agaricus blazei-induced human leukemic U937 cell apoptosis through dephoshorylation of Akt.

    PubMed

    Jin, Cheng-Yun; Moon, Dong-Oh; Choi, Yung Hyun; Lee, Jae-Dong; Kim, Gi-Young

    2007-08-01

    Agaricus blazei is a medicinal mushroom that possesses antimetastatic, antitumor, antimutagenic, and immunostimulating effects. However, the molecular mechanisms involved in A. blazei-mediated apoptosis remain unclear. In the present study, to elucidate the role of the Bcl-2 in A. blazei-mediated apoptosis, U937 cells were transfected with either empty vector (U937/vec) or vector containing cDNA encoding full-length Bcl-2 (U937/Bcl-2). As compared with U937/vec, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment of U937/vec with 1.0-4.0 mg/ml of A. blazei extract (ABE) for 24 h resulted in a significant induction of morphologic features indicative of apoptosis. In contrast, U937/Bcl-2 exposed to the same ABE treatment only exhibited a slight induction of apoptotic features. ABE-induced apoptosis was accompanied by downregulation of antiapoptotic proteins such as X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein (cIAP)-2 and Bcl-2, activation of caspase-3, and cleavage of poly(ADP-ribose)polymerase (PARP). Ectopic expression of Bcl-2 was associated with significantly induced expression of antiapoptotic proteins, such as cIAP-2 and Bcl-2, but not XIAP. Ectopic expression of Bcl-2 also reduced caspase-3 activation and PARP cleavage in ABE treated U937 cells. Furthermore, treatment with the caspase-3 inhibitor z-DEVD-fmk was sufficient to restore cell viability following ABE treatment. This increase in viability was ascribed to downregulation of caspase-3 and blockage of PARP and PLC-gamma cleavage. ABE also triggered the downregulation of Akt, and combined treatment with LY294002 (an inhibitor of Akt) significantly decreased cell viability. The results indicated that major regulators of ABE-induced apoptosis in human leukemic U937 cells are Bcl-2 and caspase-3, which are associated with dephosphorylation of the Akt signal pathway.

  9. The Enigmatic Roles of Caspases in Tumor Development

    PubMed Central

    Jäger, Richard; Zwacka, Ralf M.

    2010-01-01

    One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may

  10. Induction of apoptosis by sarijang, a bamboo salt sauce, in U937 human leukemia cells through the activation of caspases

    PubMed Central

    CHOI, EUN-A; PARK, CHEOL; HAN, MIN-HO; LEE, JUN HYUK; KIM, GI-YOUNG; CHOI, BYUNG TAE; CHOI, YUNG HYUN

    2013-01-01

    Sarijang is a bamboo salt soy sauce, containing extracts of Rhynchosia nulubilis, sulfur-fed duck, dried bark of Ulmus davidiana and Allium sativum, which has been demonstrated to exert anti-inflammatory and antitumor activity. However, the cellular and molecular mechanisms of action of sarijang have not yet been elucidated. In the present study, we investigated the pro-apoptotic effects of sarijang in an in vitro U937 human leukemia cell model. Treatment with sarijang resulted in a concentration-dependent growth inhibition of the cells, coupled with the characteristic morphological features of apoptosis. The induction of the apoptotic cell death of the U937 cells by sarijang exhibited a correlation with the upregulation of death receptor 4 (DR4), the downregulation of members of the inhibitor of apoptosis protein (IAP) family, including survivin and cellular IAP (cIAP)-1, and the cleavage of Bid. Apoptosis-inducing concentrations of sarijang also induced the activation of caspases (caspase-3, -8 and -9), accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase, β-catenin and phospholipase C-γ1. However, the apoptosis induced by sarijang was significantly inhibited by z-VED-fmk, a pan-caspase inhibitor, which demonstrated the importance of caspases in the process. These results suggested that sarijang may be a potential chemotherapeutic agent for use in the control of U937 human leukemia cells. Further studies are required to identify the active compounds in sarijang. PMID:24137193

  11. Inhibition of Histone Deacetylases Permits Lipopolysaccharide-Mediated Secretion of Bioactive IL-1β via a Caspase-1-Independent Mechanism.

    PubMed

    Stammler, Dominik; Eigenbrod, Tatjana; Menz, Sarah; Frick, Julia S; Sweet, Matthew J; Shakespear, Melanie R; Jantsch, Jonathan; Siegert, Isabel; Wölfle, Sabine; Langer, Julian D; Oehme, Ina; Schaefer, Liliana; Fischer, Andre; Knievel, Judith; Heeg, Klaus; Dalpke, Alexander H; Bode, Konrad A

    2015-12-01

    Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1β processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1β maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1β secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1β, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1β cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1β by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1β, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications. Copyright © 2015 by The American Association of Immunologists, Inc.

  12. Interaction between endoplasmic reticulum stress and caspase 8 activation in retrovirus MoMuLV-ts1-infected astrocytes.

    PubMed

    Liu, Na; Scofield, Virginia L; Qiang, Wenan; Yan, Mingshan; Kuang, Xianghong; Wong, Paul K Y

    2006-05-10

    The murine retrovirus, MoMuLV-ts1, induces progressive paralysis and immune deficiency in FVB/N mice. We have reported previously that ts1 infection causes apoptosis in astrocytes via endoplasmic reticulum (ER) and mitochondrial stress (Liu, N., Kuang, X., Kim, H.T., Stoica, G., Qiang, W., Scofield, V.L., Wong, P.K.Y. Wong. 2004. Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in MoMuLV-ts1-induced apoptosis in astrocytes. J. NeuroVirol. 10, 189-198). In the present study, we show that caspase 8 activation in these cells is mediated through ER stress-associated elevation of death receptor DR5 and the C/EBP homologous protein (GADD153/CHOP), an ER stress-initiated transcription factor, rather than through TNFalpha and TNF-R1 interactions on the cell surface. Treatment with Z-IETD-FMK, a specific inhibitor of caspase 8 enzymatic activity, reduced ER stress by two mechanisms: by inhibiting caspase 8 activation, and by preventing cleavage of the ER-associated membrane protein BAP31 into BAP20, which exacerbates the ER stress response. These findings suggest that caspase 8- and ER stress-associated apoptotic pathways are linked in ts1-infected astrocytes.

  13. BDNF protects neurons following injury by modulation of caspase activity.

    PubMed

    Kim, Dong H; Zhao, Xiurong

    2005-01-01

    Neurotrophins can protect against apoptotic death following neuronal injury. In a previous article, we showed that activation of the trk receptor is required, but the subsequent mechanisms of action remain unclear. Because the caspase family of cysteine proteases plays a central role in the apoptotic process, we examined the effect of the neurotrophins on caspase activation. Primary neuronal cultures from the embryonic rat cortex were injured with radiation, oxygen deprivation, or oxygen-glucose deprivation. Neurons were treated with brain-derived growth factor (BDNF) or caspase inhibitors. The level of injury was assayed by measuring lactate dehydrogenase release. Western blots were used to note the presence and activation of the caspases 1, 2, 3, 8, and 9--with and without treatment with BDNF. Proenzymes for caspases 1, 2, and 3--but not for caspases 8 or 9 were expressed. With radiation or oxygen deprivation, but not oxygen-glucose deprivation, caspase 3 was activated. Treatment with BDNF was protective against radiation and oxygen deprivation only. Treatment with BDNF also blocked the activation of caspase 3. A similar effect was achieved by directly blocking caspase 1 or 3 activation using an inhibitor. In this study, we showed that BDNF treatment inhibits caspase 3 activation following neuronal injury. This is a central event: when injury did not lead to caspase 3 activation, BDNF treatment was not protective. These results suggest one mechanism by which the neurotrophins protect neurons following injury.

  14. The role of caspase-2 in stress-induced apoptosis

    PubMed Central

    Bouchier-Hayes, Lisa

    2010-01-01

    Abstract Caspase-2 is the most evolutionarily conserved of all the caspases, yet it has a poorly defined role in apoptotic pathways. This is mainly due to a dearth of techniques to determine the activation status of caspase-2 and the lack of an abnormal phenotype in caspase-2 deficient mice. Nevertheless, emerging evidence suggests that caspase-2 may have important functions in a number of stress-induced cell death pathways, in cell cycle maintenance and regulation of tumour progression. This review discusses recent advances that have been made to help elucidate the true role of this elusive caspase and the potential contribution of caspase-2 to the pathology of human diseases including cancer. PMID:20158568

  15. Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

    PubMed Central

    Heise, Christopher E.; Murray, Jeremy; Augustyn, Katherine E.; Bravo, Brandon; Chugha, Preeti; Cohen, Frederick; Giannetti, Anthony M.; Gibbons, Paul; Hannoush, Rami N.; Hearn, Brian R.; Jaishankar, Priyadarshini; Ly, Cuong Q.; Shah, Kinjalkumar; Stanger, Karen; Steffek, Micah; Tang, Yinyan; Zhao, Xianrui; Lewcock, Joseph W.; Renslo, Adam R.; Flygare, John; Arkin, Michelle R.

    2012-01-01

    Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound’s inhibitory activity is also dependent on the amino acid sequence and P1’ character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity. PMID:23227217

  16. ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication

    PubMed Central

    Hida, Shigeaki; Fujii, Chifumi; Taniguchi, Shun’ichiro; Ito, Kensuke; Matsumura, Tomio; Okada, Nagisa; Sakaizawa, Takashi; Kobayashi, Akira; Takeoka, Michiko; Miyagawa, Shin-ichi

    2017-01-01

    ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy. PMID:28056049

  17. Glucosidase II β-subunit, a novel substrate for caspase-3-like activity in rice, plays as a molecular switch between autophagy and programmed cell death

    PubMed Central

    Cui, Jing; Chen, Bing; Wang, Hongjuan; Han, Yue; Chen, Xi; Zhang, Wei

    2016-01-01

    Endoplasmic reticulum (ER) stress activates unfolded protein response (UPR) and autophagy. However, prolonged, severe stresses activate programmed cell death (PCD) in both animal and plant cells. Compared to the well-studied UPR pathway, the molecular mechanisms of ER-stress-induced PCD are less understood. Here, we report the identification of Gas2, the glucosidase II β subunit in the ER, as a potential switch between PCD and autophagy in rice. MS analysis identified Gas2, GRP94, and HSP40 protein in a purified caspase-3-like activity from heat stressed rice cell suspensions. The three corresponding genes were down-regulated under DTT-induced ER stress. Gas2 and GRP94 were localized to the ER, while HSP40 localized to the cytoplasm. Compared to wild-type, a Gas2 RNAi cell line was much sensitive to DTT treatment and had high levels of autophagy. Both caspase-3 and heat-stressed cell suspension lysate could cleave Gas2, producing a 14 kDa N-terminal fragment. Conditional expression of corresponding C-terminal fragment resulted in enhanced caspase-3-like activity in the protoplasts under heat stress. We proposed that mild ER stress causes down-regulation of Gas2 and induces autophagy, while severe stress results in Gas2 cleavage by caspase-3-like activity and the cleavage product amplifies this activity, possibly participating in the initiation of PCD. PMID:27538481

  18. Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

    PubMed

    Newell, Jennifer L; Fay, Philip J

    2009-04-24

    The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

  19. Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses.

    PubMed

    Bulau, Ana-Maria; Nold, Marcel F; Li, Suzhao; Nold-Petry, Claudia A; Fink, Michaela; Mansell, Ashley; Schwerd, Tobias; Hong, Jaewoo; Rubartelli, Anna; Dinarello, Charles A; Bufler, Philip

    2014-02-18

    IL-37 is a fundamental inhibitor of innate immunity. Human IL-37 has a caspase-1 cleavage site and translocates to the nucleus upon LPS stimulation. Here, we investigated whether caspase-1 processing affects IL-37-mediated suppression of LPS-induced cytokines and the release from cells by analyzing a caspase-1 cleavage site mutant IL-37 (IL-37D20A). Nuclear translocation of IL-37D20A is significantly impaired compared with WT IL-37 in transfected cells. LPS-induced IL-6 was decreased in cells expressing WT IL-37 but not IL-37D20A. The function of IL-37 in transfected bone marrow-derived macrophages is nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-dependent, because IL-37 transfection in apoptosis-associated speck-like protein containing a carboxyl-terminal caspase recruitment domain- and NLRP3-deficient cells does not reduce levels of IL-6 and IL-1β upon LPS stimulation. IL-37-expressing macrophages release both precursor and mature IL-37, but only the externalization of mature IL-37 was dependent on ATP. Precursor and mature IL-37 was also secreted from human dendritic cells and peripheral blood mononuclear cells. To determine whether IL-37 is active in the extracellular compartment, we pretreated IL-37 transgenic mice with IL-37-neutralizing antibodies before LPS challenge. In IL-37-expressing mice, neutralizing IL-37 antibodies reversed the suppression of LPS-induced serum IL-6. In contrast, the addition of neutralizing antibody did not reverse suppression of LPS-induced IL-6 in mouse macrophages transfected with IL-37. Although caspase-1 is required for nuclear translocation of intracellular IL-37 and for secretion of mature IL-37, the release of the IL-37 precursor is independent of caspase-1 activation. IL-37 now emerges as a dual-function cytokine with intra- and extracellular properties for suppressing innate inflammation.

  20. Application of a FRET probe for Caspase-3 activation in living HeLa cells by sequentially treated cisplatin and TRAIL

    NASA Astrophysics Data System (ADS)

    Lin, Juqiang; Zhang, Zhihong; Yi, Qiushi; Zeng, Shaoqun; Luo, Qingming

    2006-02-01

    Caspase-3 is a kind of cysteine proteases that plays an important role in cell apoptosis. We have constructed a FRET (fluorescence resonance energy transfer) probe fused with ECFP (enhanced cyan fluorescence protein) and DsRed (Discosoma red fluorescent protein) with a linker containing a caspase-3 cleavage sequence (CCS, DEVD).It could be observed much change in fluorescence emission ratio when the probe was cleaved by caspase-3. Therefore, application of this probe we can real-time detected the activation of caspase-3. It was already confirmed that caspase-3 was activated in HeLa cells treated by cisplatin or TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand). In the present study, we detected the activation of caspase-3 during cisplatin or TRAIL induced apoptosis in living HeLa cells, and also observed the activation of caspase-3 caused by both cisplatin and TRAIL combined treatment. Our results demonstrated a synergistic effect between cisplatin and TRAIL. Cisplatin is one of the most broadly used drugs in the Clinical applications of cancer chemotherapy, and TRAIL, which belongs to the TNF family proteins, can selectively induce apoptosis in many transformed cells but not in normal cells. Therefore, TRAIL is a very valuably prospective utility as its potential tumor-specific cancer therapeutic. Most of anticancer drugs can induce apoptosis which mediated by the activation of caspase pathway. We can select the best synergistic effect group by our FRET probe. This finding would be useful in the design of treatment modalities for patients.

  1. p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3*

    PubMed Central

    Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P.; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T.; Arumäe, Urmas; Lindholm, Dan

    2016-01-01

    Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. PMID:26984409

  2. Caspase-14 protects against epidermal UVB photodamage and water loss.

    PubMed

    Denecker, Geertrui; Hoste, Esther; Gilbert, Barbara; Hochepied, Tino; Ovaere, Petra; Lippens, Saskia; Van den Broecke, Caroline; Van Damme, Petra; D'Herde, Katharina; Hachem, Jean-Pierre; Borgonie, Gaetan; Presland, Richard B; Schoonjans, Luc; Libert, Claude; Vandekerckhove, Joël; Gevaert, Kris; Vandenabeele, Peter; Declercq, Wim

    2007-06-01

    Caspase-14 belongs to a conserved family of aspartate-specific proteinases. Its expression is restricted almost exclusively to the suprabasal layers of the epidermis and the hair follicles. Moreover, the proteolytic activation of caspase-14 is associated with stratum corneum formation, implicating caspase-14 in terminal keratinocyte differentiation and cornification. Here, we show that the skin of caspase-14-deficient mice was shiny and lichenified, indicating an altered stratum-corneum composition. Caspase-14-deficient epidermis contained significantly more alveolar keratohyalin F-granules, the profilaggrin stores. Accordingly, caspase-14-deficient epidermis is characterized by an altered profilaggrin processing pattern and we show that recombinant caspase-14 can directly cleave profilaggrin in vitro. Caspase-14-deficient epidermis is characterized by reduced skin-hydration levels and increased water loss. In view of the important role of filaggrin in the structure and moisturization of the skin, the knockout phenotype could be explained by an aberrant processing of filaggrin. Importantly, the skin of caspase-14-deficient mice was highly sensitive to the formation of cyclobutane pyrimidine dimers after UVB irradiation, leading to increased levels of UVB-induced apoptosis. Removal of the stratum corneum indicate that caspase-14 controls the UVB scavenging capacity of the stratum corneum.

  3. PARP-1 regulates the expression of caspase-11

    SciTech Connect

    Yoo, Lang; Hong, Seokheon; Shin, Ki Soon; Kang, Shin Jung

    2011-05-13

    Highlights: {yields} Knockdown of PARP-1 suppresses the LPS-induced expression of caspase-11. {yields} Knockdown of PARP-1 suppresses the caspase-11 promoter activity following LPS stimulation. {yields} PARP-1 is recruited to the caspase-11 promoter region containing NF-{kappa}B-binding sites following LPS stimulation. {yields} PARP-1 inhibitors cannot suppress the caspase-11 induction. {yields} PARP-1 does not suppress IFN-{gamma}-induced expression of caspase-11. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) is a multifunctional enzyme that regulates DNA repair, cell death and transcription of inflammatory proteins. In the present study, we present evidence that PARP-1 regulates the expression of caspase-11 following lipopolysaccharide (LPS) stimulation. Knockdown of PARP-1 suppressed the LPS-induced expression of caspase-11 at both mRNA and protein levels as well as caspase-11 promoter activity. Importantly, PARP-1 was recruited to the caspase-11 promoter region containing predicted nuclear factor (NF)-{kappa}B-binding sites when examined by chromatin immunoprecipitation assay. However, knockdown of PARP-1 did not suppress the expression of caspase-11 induced by interferon-{gamma} that activates signal transducer and activator of transcription 1 but not NF-{kappa}B. PARP-1 enzymatic activity was not required for the caspase-11 upregulation since pharmacological inhibitors of PARP-1 did not suppress the induction of caspase-11. Our results suggest that PARP-1, as a transcriptional cofactor for NF-{kappa}B, regulates the induction of caspase-11 at a transcriptional level.

  4. Apoptosis and age-related disorders: role of caspase-dependent and caspase-independent pathways.

    PubMed

    Nicotera, Pierluigi

    2002-02-28

    The execution of the apoptotic program involves a relatively limited number of pathways that converge on the activation of the caspase family of proteases. However, there is increasing evidence that other protease families may contribute to produce apoptotic-like features. This has posed the question as to whether caspase inhibitors may then be used to treat diseases characterised by an excess apoptosis. In several neurodegenerative diseases including acute neuronal loss as in stroke or slowly developing diseases at least two major events contribute to neurodegeneration: the loss of neuronal connectivity and cell loss. In many of these conditions, mitochondrial dysfunction and the resulting ATP depletion may preclude caspase activation, and consequently switch execution of cell death towards necrosis. A block or partial inhibition of the typical apoptotic demise may have profound implications in vivo, as persistence within the nervous system of damaged, but 'undead' cells, followed by delayed lysis may favour neuroinflammatory reactions. Furthermore, caspases may be involved in loss of neurons, but not in the loss of connectivity that seems to initiate degenerative processes in the nervous system. Some recent findings, which suggest that degenerating neurons may use multiple execution pathways will be discussed.

  5. A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3.

    PubMed

    Weber, Jonasz J; Golla, Matthias; Guaitoli, Giambattista; Wanichawan, Pimthanya; Hayer, Stefanie N; Hauser, Stefan; Krahl, Ann-Christin; Nagel, Maike; Samer, Sebastian; Aronica, Eleonora; Carlson, Cathrine R; Schöls, Ludger; Riess, Olaf; Gloeckner, Christian J; Nguyen, Huu P; Hübener-Schmid, Jeannette

    2017-03-08

    Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes

    PubMed Central

    Aglietti, Robin A.; Estevez, Alberto; Gupta, Aaron; Ramirez, Monica Gonzalez; Liu, Peter S.; Kayagaki, Nobuhiko; Ciferri, Claudio; Dixit, Vishva M.; Dueber, Erin C.

    2016-01-01

    Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca2+ from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane. PMID:27339137

  7. Drosophila Spaghetti and Doubletime Link the Circadian Clock and Light to Caspases, Apoptosis and Tauopathy

    PubMed Central

    Means, John C.; Venkatesan, Anandakrishnan; Gerdes, Bryan; Fan, Jin-Yuan; Bjes, Edward S.; Price, Jeffrey L.

    2015-01-01

    While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes

  8. Therapeutic strategies targeting caspase inhibition following spinal cord injury in rats.

    PubMed

    Ozawa, Hiroshi; Keane, Robert W; Marcillo, Alexander E; Diaz, Paulo H; Dietrich, W Dalton

    2002-09-01

    Apoptosis-modulating therapeutics using active-site mimetic peptide ketones (z-VAD-fluoromethylketone (fmk)) have been reported to be efficacious in delaying the apoptotic response in central nervous system lesions. The purpose of the present study was to examine whether the caspase inhibitor z-VAD fmk prevents apoptosis and improves neurological deficit and tissue damage. One-hundred twenty female Sprague-Dawley rats were randomized into groups that were administered 25 microg of z-VAD-fmk or vehicle 30 min and 24 h after moderate spinal cord contusion (NYU impactor, 12.5 mm at T10). Several routes of administration were tested: (1) via Gelfoam placed on the spinal cord, (2) into the cisterna magna via a subarachnoidal catheter, (3) intravenously via the external jugular vein, or (4) intraperitoneally. Another group was injected with 50 microg of zVAD-fmk or vehicle intraperitoneally 30 min, 24, 48, and 72 h after injury. Animals were evaluated for locomotor function (BBB score) at weekly intervals for 6 weeks after injury and treatment. Spinal cords were then processed for histological analysis to determine whether zVAD-fmk treatment decreased contusion volume. Other spinal cord samples were harvested 24 h after injury and examined for cleavage of XIAP by immunoblot analysis. There were no significant differences in the BBB scores, contusion volumes, and XIAP cleavage between animals receiving the broad specific caspase inhibitor by the various routes and animals receiving vehicle alone. These findings raise critical questions about the use of peptide ketone apoptotic inhibitors in improving functional and histopathological outcomes following spinal cord injury.

  9. Does Cleavage Work at Work? Men, but Not Women, Falsely Believe Cleavage Sells a Weak Product

    ERIC Educational Resources Information Center

    Glick, Peter; Chrislock, Karyna; Petersik, Korinne; Vijay, Madhuri; Turek, Aleksandra

    2008-01-01

    We examined whether men, but not women, would be distracted by a female sales representative's exposed cleavage, leading to greater perceived efficacy for a weak, but not for a strong product. A community sample of 88 men and 97 women viewed a video of a female pharmaceutical sales representative who (a) had exposed cleavage or dressed modestly…

  10. Does Cleavage Work at Work? Men, but Not Women, Falsely Believe Cleavage Sells a Weak Product

    ERIC Educational Resources Information Center

    Glick, Peter; Chrislock, Karyna; Petersik, Korinne; Vijay, Madhuri; Turek, Aleksandra

    2008-01-01

    We examined whether men, but not women, would be distracted by a female sales representative's exposed cleavage, leading to greater perceived efficacy for a weak, but not for a strong product. A community sample of 88 men and 97 women viewed a video of a female pharmaceutical sales representative who (a) had exposed cleavage or dressed modestly…

  11. Selective cleavage of pepsin by molybdenum metallopeptidase

    SciTech Connect

    Yenjai, Sudarat; Malaikaew, Pinpinat; Liwporncharoenvong, Teerayuth; Buranaprapuk, Apinya

    2012-03-02

    Graphical abstract: Molybdenum metallopeptidase: the Mo(VI) cluster with six molybdenum cations has the ability to cleave protein under mild conditions (37 Degree-Sign C, pH 7) without reducing agents. The reaction required only low concentration of ammonium heptamolybdatetetrahydrate ((NH{sub 4}){sub 6}Mo{sub 7}O{sub 24}{center_dot}4H{sub 2}O) (0.125 mM). The reaction undergoes possibly via a hydrolytic mechanism. This is the first demonstration of protein cleavage by a molybdenum cluster. Highlights: Black-Right-Pointing-Pointer This is the first demonstration of protein cleavage by a Mo(VI) cluster with six molybdenum cations. Black-Right-Pointing-Pointer The cleavage reaction undergoes at mild conditions. Black-Right-Pointing-Pointer No need of reducing agents. Black-Right-Pointing-Pointer Only low concentration of Mo(VI) cluster and short time of incubation are needed. -- Abstract: In this study, the cleavage of protein by molybdenum cluster is reported for the first time. The protein target used is porcine pepsin. The data presented in this study show that pepsin is cleaved to at least three fragments with molecular weights of {approx}23, {approx}19 and {approx}16 kDa when the mixture of the protein and ammonium heptamolybdate tetrahydrate ((NH{sub 4}){sub 6}Mo{sub 7}O{sub 24}{center_dot}4H{sub 2}O) was incubated at 37 Degree-Sign C for 24 h. No self cleavage of pepsin occurs at 37 Degree-Sign C, 24 h indicating that the reaction is mediated by the metal ions. N-terminal sequencing of the peptide fragments indicated three cleavage sites of pepsin between Leu 112-Tyr 113, Leu 166-Leu 167 and Leu 178-Asn 179. The cleavage reaction occurs after incubation of the mixture of pepsin and (NH{sub 4}){sub 6}Mo{sub 7}O{sub 24}{center_dot}4H{sub 2}O) only for 2 h. However, the specificity of the cleavage decreases when incubation time is longer than 48 h. The mechanism for cleavage of pepsin is expected to be hydrolytic chemistry of the amide bonds in the protein

  12. Senoptosis: non-lethal DNA cleavage as a route to deep senescence.

    PubMed

    Studencka, Maja; Schaber, Jörg

    2017-05-09

    DNA-damage-induced apoptosis and cellular senescence are perceived as two distinct cell fates. We found that after ionizing radiation (IR)-induced DNA damage the majority (up to 70 %) of senescent human diploid fibroblasts (HDFs) were subjected to controlled cleavage of DNA, resulting in the establishment of a viable and stable sub-G1 population, i.e. deeply senescent cells. We show that in senescent HDFs this DNA cleavage is triggered by modest loss of the mitochondrial membrane potential, which is not sufficient to activate caspases, but strong enough to release mitochondrial endonuclease G (EndoG). We demonstrate that upon γ-irradiation in HDFs EndoG translocates into the nucleus playing an essential role in the non-lethal cleavage of damaged DNA. Notably, the established sub-G1 cell population does not contribute to the senescence-associated secretory phenotype (SASP), however, it exhibits increased senescence-associated β-galactosidase activity. We show that EndoG knockdown causes an increase in DNA damage, indicating a role of this enzyme in DNA repair. Thus, we conclude that IR-induced deep senescence of HDFs exhibits features of both senescence, such as cell cycle arrest and viability, and apoptosis like reduced DNA content and no SASP, and, resembles uncomplete or stalled apoptosis, a phenomenon we term senoptosis.

  13. Structural basis of cohesin cleavage by separase

    PubMed Central

    Lin, Zhonghui; Luo, Xuelian; Yu, Hongtao

    2016-01-01

    Accurate chromosome segregation requires timely dissolution of chromosome cohesion after chromosomes are properly attached to the mitotic spindle. Separase is absolutely essential for cohesion dissolution in organisms from yeast to man1,2. It cleaves the kleisin subunit of cohesin and opens the cohesin ring to allow chromosome segregation. Cohesin cleavage is spatiotemporally controlled by separase-associated regulatory proteins, including the inhibitory chaperone securin3–6, and by phosphorylation of both the enzyme and substrates7–12. Dysregulation of this process causes chromosome missegregation and aneuploidy, contributing to cancer and birth defects. Despite its essential functions, atomic structures of separase have not been determined. Here, we report crystal structures of the separase protease domain from Chaetomium thermophilum, alone or covalently bound to unphosphorylated and phosphorylated inhibitory peptides derived from a cohesin cleavage site. These structures reveal how separase recognizes cohesin and how cohesin phosphorylation by polo-like kinase 1 (Plk1) enhances cleavage. Consistent with a previous cellular study13, mutating two securin residues in a conserved motif that partially matches the separase cleavage consensus converts securin from a separase inhibitor to a substrate. Our study establishes atomic mechanisms of substrate cleavage by separase and suggests competitive inhibition by securin. PMID:27027290

  14. Caspase Deficiency: Involvement in Breast Carcinogenesis and Resistance

    DTIC Science & Technology

    2000-07-01

    sterol-regulatory element-binding protein (SREBPs)(19), gelsolin (20), the Ul-associated 70 kDa protein (21), D4-GDI (22), DNA fragmentation factor...3C), the observed proteolysis was a consequence of caspase 3 reconstitution. Caspase 3 was required for doxorubicin induced DNA fragmentation DNA ... fragmentation is a key feature associated with apoptosis (6). Caspase 3 is required for DNA fragmentation in tumor necrosis factor-a (TNF-cc) induced

  15. Nonapoptotic Function of Caspase-6 in Promoting Mammary Carcinogenesis

    DTIC Science & Technology

    2012-01-01

    Figure 4), normal human mammary tissues show little caspase-6 staining and about 60% of cancer tissues showed strong caspase-6 staining (Figure 4...Center) were stained for caspse-6. The included one of the three normal mammary tissues (upper panels) contains little caspase-6 staining . The cancer...lower panel) is highly positive of caspse-6 staining . A 100X magnification (left panels) and a 400X magnification are shown. About 60% of

  16. Involvement of p21Waf1/Cip1 cleavage during roscovitine-induced apoptosis in non-small cell lung cancer cells.

    PubMed

    Zhang, Tao; Jiang, Tao; Zhang, Feng; Li, Chen; Zhou, Yong-An; Zhu, Yi-Fang; Li, Xiao-Fei

    2010-01-01

    Roscovitine, a cyclin-dependent kinases (CDKs) inhibitor, has been reported to have anti-tumor effects in some cancer cell lines by inducing apoptosis. However, the exact underlying mechanisms are not fully understood. Here, we report that roscovitine induces expression and cleavage of the universal CDK inhibitor p21Waf1/Cip1 in non-small cell lung cancer (NSCLC) A549 cells in a dose-dependent manner. Western blots of roscovitine-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band that was not detected in control cultures. CDK2 activity and PCNA expression were repressed with increasing dose of roscovitine. Accompanying these molecular changes was a progressive arrest of G2 phase and decreasing of 5-bromo-2-deoxyuridine (Brdu) incorporation of S phase cells. Caspase-3 inhibitor z-DEVD-fmk almost completely abolished roscovitine-induced apoptosis, as well as the appearance of 15 kDa band, indicating that p21Waf1/Cip1 cleavage was mediated by caspase-3 activity. Furthermore, this band was predominant in the floating apoptotic cells, while weakened in the adherent cells which were vital and pre-apoptotic. We also showed that roscovitine induced an enhanced expression of gamma-H2AX, which was blocked by caspase-3 inhibition, suggesting that p21Waf1/Cip1 cleavage may interfere with DNA repair, leading to increased frequency of double strand breaks (DSBs) and enhanced apoptosis. Here we show, for the first time, that p21Waf1/Cip1 cleavage, which is mediated by caspase-3 activity, is involved in roscovitine-induced apoptosis.

  17. δ-Cadinene inhibits the growth of ovarian cancer cells via caspase-dependent apoptosis and cell cycle arrest.

    PubMed

    Hui, Li-Mei; Zhao, Guo-Dong; Zhao, Jian-Jun

    2015-01-01

    Ovarian cancer is one of the most common causes of mortality among all cancers in females and is the primary cause of mortality from gynecological malignancies. The objective of the current research work was to evaluate a naturally occurring sesquiterpene-δ-Cadinene for its antiproliferative and apoptotic effects on human ovary cancer (OVCAR-3) cells. We also demonstrated the effect of δ-Cadinene on cell cycle phase distribution, intracellular damage and caspase activation. Sulforhodamine B (SRB) assay was used to evaluate the antiproliferative effect of δ-cadinene on OVCAR-3 cells. Cellular morphology after δ-cadinene treatment was demonstrated by inverted phase contrast microscopy, fluorescence microscopy and transmission electron microscopy. Flow cytometry was used to analyze the effect of δ-cadinene on cell cycle phase distribution and apoptosis using propidium iodide and Annexin V-fluorescein isothiocyanate (FITC)/PI kit. The results revealed that δ-cadinene induced dose-dependent as well as time-dependent growth inhibitory effects on OVACR-3 cell line. δ-cadinene also induced cell shrinkage, chromatin condensation and nuclear membrane rupture which are characteristic of apoptosis. Treatment with different doses of δ-cadinene also led to cell cycle arrest in sub-G1 phase which showed dose-dependence. Western blotting assay revealed that δ-cadinene led to activation of caspases in OVCAR-3 cancer cells. PARP cleavage was noticed at 50 µM dose of δ-cadinene with the advent of the cleaved 85-kDa fragment after exposure to δ-cadinene. At 100 µM, only the cleaved form of PARP was detectable. Pro-caspase-8 expression remained unaltered until 10 µM dose of δ-cadinene. However, at 50 and 100 µM dose, pro-caspase-8 expression was no longer detectable. There was a significant increase in the caspase-9 expression levels after 50 and 100 µM δ-cadinene treatments.

  18. Molecular cloning and characterization of the first caspase in the Striped Stem Borer, Chilo suppressalis.

    PubMed

    Lu, Ming-Xing; Du, Yu-Zhou; Cao, Shuang-Shuang; Liu, Pingyang; Li, Jianyong

    2013-05-15

    Apoptosis is executed through the activity of the caspases that are aspartyl-specific proteases. In this study, we isolated the caspase gene (Cscaspase-1) of Chilo suppressalis (one of the leading pests responsible for destruction of rice crops). It possesses the open reading frame (ORF) of 295 amino acids including prodomain, large subunit and small subunits, and two cleavage sites (Asp23 and Asp194) were found to be located among them. In addition to these profiles, Cscaspase-1 contains two active sites (His134 and Cys176). Genomic analysis demonstrated there was no intron in the genome of Cscaspase-1. The Cscaspase-1 transcripts were found in all tissues of the fifth instar larvae, and higher levels were found in the midgut, hindgut and Malpighian tubules. Examination of Cscaspase-1 expression in different developmental stages indicated low constitutive levels in the eggs and early larvae stages, and higher abundances were exhibited in the last larvae and pupae stages. The relative mRNA levels of Cscaspase-1 were induced by heat and cold temperatures. For example, the highest increase of Cscaspase-1 transcription was at -3 °C and 36 °C respectively. In a word, Cscaspase-1 plays a role of effector in the apoptosis of C. suppressalis. It also correlates with development, metamorphosis and thermotolerance of C. suppreassalis.

  19. Apoptosis induction of human lung carcinoma cells by Chan Su (Venenum Bufonis) through activation of caspases.

    PubMed

    Yun, Hye Ri; Yoo, Hwa Seung; Shin, Dong Yeok; Hong, Su Hyun; Kim, Jong-Hwan; Cho, Chong Kwan; Choi, Yung Hyun

    2009-09-01

    Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads, and has been used as an oriental drug for the treatment of a number of diseases, including cancer. In this study, the potential of Chan Su (skin of Venenum Bufonis) to induce apoptosis in human lung carcinoma A549 cells was investigated. Treatment of A549 cells with skin of Venenum Bufonis resulted in the inhibition of cell growth and viability and the induction of apoptosis, which was shown by trypan blue counts, MTT assay, DAPI staining and flow cytometry analysis. The increase in apoptosis that was induced by skin of Venenum Bufonis was correlated with down-regulation of anti-apoptotic Bcl-2 expression, up-regulation of pro-apoptotic Fas ligand and death receptor 4, and a decrease in the mitochondrial membrane potential. Skin of Venenum Bufonis treatment induced the proteolytic activation of caspases and a concomitant degradation of poly(ADP-ribose)-polymerase and beta-catenin protein. Cleavage of Bid and a down-regulation of the inhibitor of apoptosis family proteins were also observed in skin of Venenum Bufonis-treated A549 cells. Data from this study indicates that SVB induces the apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic and mitochondria-mediated intrinsic caspase pathways.

  20. Momordica charantia Extract Induces Apoptosis in Human Cancer Cells through Caspase- and Mitochondria-Dependent Pathways

    PubMed Central

    Li, Chia-Jung; Tsang, Shih-Fang; Tsai, Chun-Hao; Tsai, Hsin-Yi; Chyuan, Jong-Ho; Hsu, Hsue-Yin

    2012-01-01

    Plants are an invaluable source of potential new anti-cancer drugs. Momordica charantia is one of these plants with both edible and medical value and reported to exhibit anticancer activity. To explore the potential effectiveness of Momordica charantia, methanol extract of Momordica charantia (MCME) was used to evaluate the cytotoxic activity on four human cancer cell lines, Hone-1 nasopharyngeal carcinoma cells, AGS gastric adenocarcinoma cells, HCT-116 colorectal carcinoma cells, and CL1-0 lung adenocarcinoma cells, in this study. MCME showed cytotoxic activity towards all cancer cells tested, with the approximate IC50 ranging from 0.25 to 0.35 mg/mL at 24 h. MCME induced cell death was found to be time-dependent in these cells. Apoptosis was demonstrated by DAPI staining and DNA fragmentation analysis using agarose gel electrophoresis. MCME activated caspase-3 and enhanced the cleavage of downstream DFF45 and PARP, subsequently leading to DNA fragmentation and nuclear condensation. The apoptogenic protein, Bax, was increased, whereas Bcl-2 was decreased after treating for 24 h in all cancer cells, indicating the involvement of mitochondrial pathway in MCME-induced cell death. These findings indicate that MCME has cytotoxic effects on human cancer cells and exhibits promising anti-cancer activity by triggering apoptosis through the regulation of caspases and mitochondria. PMID:23091557

  1. Caspase-9 mediates Puma activation in UCN-01-induced apoptosis.

    PubMed

    Nie, C; Luo, Y; Zhao, X; Luo, N; Tong, A; Liu, X; Yuan, Z; Wang, C; Wei, Y

    2014-10-30

    The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.

  2. Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases.

    PubMed

    Lüthi, Alexander U; Cullen, Sean P; McNeela, Edel A; Duriez, Patrick J; Afonina, Inna S; Sheridan, Clare; Brumatti, Gabriela; Taylor, Rebecca C; Kersse, Kristof; Vandenabeele, Peter; Lavelle, Ed C; Martin, Seamus J

    2009-07-17

    Interleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent proteolysis, similar to the proinflammatory cytokines IL-1 beta and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-kappaB transcription factor. Indeed, caspase-dependent proteolysis of IL-33 dramatically attenuated IL-33 bioactivity in vitro and in vivo. These data suggest that IL-33 does not require proteolysis for activation, but rather, that IL-33 bioactivity is diminished through caspase-dependent proteolysis within apoptotic cells. Thus, caspase-mediated proteolysis acts as a switch to dampen the proinflammatory properties of IL-33.

  3. Inactivating mutations of CASPASE-7 gene in human cancers.

    PubMed

    Soung, Young Hwa; Lee, Jong Woo; Kim, Hong Sug; Park, Won Sang; Kim, Su Young; Lee, Jong Heun; Park, Jik Young; Cho, Yong Gu; Kim, Chang Jae; Park, Yong Gyu; Nam, Suk Woo; Jeong, Seong Whan; Kim, Sang Ho; Lee, Jung Young; Yoo, Nam Jin; Lee, Sug Hyung

    2003-09-11

    Caspase-7 is a caspase involved in the execution phase of apoptosis. To explore the possibility that the genetic alterations of CASPASE-7 might be involved in the development of human cancers, we analysed the entire coding region and all splice sites of human CASPASE-7 gene for the detection of somatic mutations in a series of human solid cancers, including carcinomas from stomach, colon, head/neck, esophagus, urinary bladder and lung. Overall, we detected CASPASE-7 mutations in two of 98 colon carcinomas (2.0%), one of 50 esophageal carcinomas (2.0%) and one of 33 head/neck carcinomas (3.0%). We expressed the tumor-derived caspase-7 mutants in 293 T cells and found that the apoptosis was reduced compared to the wild-type caspase-7. This is the first report on the CASPASE-7 gene mutations in human malignancies, and our data suggest that the inactivating mutations of the CASPASE-7 gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human solid cancers.

  4. Ceramides stimulate caspase-14 expression in human keratinocytes

    PubMed Central

    Jiang, Yan J.; Kim, Peggy; Uchida, Yoshikazu; Elias, Peter M.; Bikle, Daniel D.; Grunfeld, Carl; Feingold, Kenneth R.

    2014-01-01

    Caspase-14 is an enzyme that is expressed predominantly in cornifying epithelia and catalyses the degradation of profilaggrin. Additionally, caspase-14 plays an important role in the terminal differentiation of keratinocytes. However, how caspase-14 expression is regulated remains largely unknown. Here we demonstrate that ceramides (C2-Cer and C6-Cer), but not other sphingolipids (C8-glucosylceramides, sphinganine, sphingosine-1-phosphate or ceramide-1-phosphate), increase caspase-14 expression (mRNA and protein) in cultured human keratinocytes in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase and ceramidase increase endogenous ceramide levels and also increase caspase-14 expression, indicating an important regulatory role for ceramides and suggesting that the conversion of ceramides to other metabolites is not required. The increase in caspase-14 expression induced by ceramides is first seen at 16 h and requires new protein synthesis, suggesting that the ceramide-induced increase is likely an indirect effect. Furthermore, ceramides increase caspase-14 gene expression primarily by increasing transcription. Blocking de novo synthesis of ceramides does not affect caspase-14 expression, suggesting that basal expression is not dependent on ceramide levels. These studies show that ceramides, an important structural lipid, stimulate caspase-14 expression providing a mechanism for coordinately regulating the formation of lipid lamellar membranes with the formation of corneocytes. PMID:23362869

  5. Nanomechanical cleavage of molybdenum disulphide atomic layers.

    PubMed

    Tang, Dai-Ming; Kvashnin, Dmitry G; Najmaei, Sina; Bando, Yoshio; Kimoto, Koji; Koskinen, Pekka; Ajayan, Pulickel M; Yakobson, Boris I; Sorokin, Pavel B; Lou, Jun; Golberg, Dmitri

    2014-04-03

    The discovery of two-dimensional materials became possible due to the mechanical cleavage technique. Despite its simplicity, the as-cleaved materials demonstrated surprising macro-continuity, high crystalline quality and extraordinary mechanical and electrical properties that triggered global research interest. Here such cleavage processes and associated mechanical behaviours are investigated by a direct in situ transmission electron microscopy probing technique, using atomically thin molybdenum disulphide layers as a model material. Our technique demonstrates layer number selective cleavage, from a monolayer to double layer and up to 23 atomic layers. In situ observations combined with molecular dynamics simulations reveal unique layer-dependent bending behaviours, from spontaneous rippling (<5 atomic layers) to homogeneous curving (~ 10 layers) and finally to kinking (20 or more layers), depending on the competition of strain energy and interfacial energy.

  6. Nanomechanical cleavage of molybdenum disulphide atomic layers

    NASA Astrophysics Data System (ADS)

    Tang, Dai-Ming; Kvashnin, Dmitry G.; Najmaei, Sina; Bando, Yoshio; Kimoto, Koji; Koskinen, Pekka; Ajayan, Pulickel M.; Yakobson, Boris I.; Sorokin, Pavel B.; Lou, Jun; Golberg, Dmitri

    2014-04-01

    The discovery of two-dimensional materials became possible due to the mechanical cleavage technique. Despite its simplicity, the as-cleaved materials demonstrated surprising macro-continuity, high crystalline quality and extraordinary mechanical and electrical properties that triggered global research interest. Here such cleavage processes and associated mechanical behaviours are investigated by a direct in situ transmission electron microscopy probing technique, using atomically thin molybdenum disulphide layers as a model material. Our technique demonstrates layer number selective cleavage, from a monolayer to double layer and up to 23 atomic layers. In situ observations combined with molecular dynamics simulations reveal unique layer-dependent bending behaviours, from spontaneous rippling (<5 atomic layers) to homogeneous curving (~ 10 layers) and finally to kinking (20 or more layers), depending on the competition of strain energy and interfacial energy.

  7. Cleavage fracture in bainitic and martensitic microstructures

    SciTech Connect

    Zhang, X.Z.; Knott, J.F.

    1999-09-29

    This paper addresses the mechanisms of cleavage fracture in the pressure-vessel steel A533B. Microstructures of single bainite microstructures exhibit a higher propensity for brittle cleavage fracture than do those of auto-tempered martensites. The K{sub 1c} values of mixed microstructures are determined by the statistical distribution of the two phases and the range of the values is bounded by limits set by those for the single-phase microstructures. The results are explained in terms of the RKR model, which involves a local cleavage stress {sigma}*{sub F} and a distance ahead of the macrocrack tip, X, as two critical parameters. It is found that the carbides or carbide colonies act as critical microcrack nuclei, and hence play a key role in determining the fracture toughness, although packet boundaries in bainite may give rise to pop-in arrests in displacement-controlled tests.

  8. Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway.

    PubMed

    Leow, San Min; Chua, Shu Xian Serene; Venkatachalam, Gireedhar; Shen, Liang; Luo, Le; Clement, Marie-Veronique

    2016-12-18

    Here we provide evidence to link sub-lethal oxidative stress to lysosomal biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of a lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1and Ctsb and Sqstm1 and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosomes biogenesis under conditions of sub-lethal oxidative stress.

  9. Neurotoxic mechanisms caused by the Alzheimer's disease-linked Swedish amyloid precursor protein mutation: oxidative stress, caspases, and the JNK pathway.

    PubMed

    Marques, Celio A; Keil, Uta; Bonert, Astrid; Steiner, Barbara; Haass, Christian; Muller, Walter E; Eckert, Anne

    2003-07-25

    Autosomal dominant forms of familial Alzheimer's disease (FAD) are caused by mutations of the amyloid precursor protein (APP) gene and by mutations of the genes encoding for presenilin 1 or presenilin 2. Simultaneously, evidence is provided that increased oxidative stress might play a crucial role in the rapid progression of the Swedish FAD. Here we investigated the effect of the Swedish double mutation (K670M/N671L) in the beta-amyloid precursor protein on oxidative stress-induced cell death mechanisms in PC12 cells. Western blot analysis and cleavage studies of caspase substrates revealed an elevated activity of the executor caspase 3 after treatment with hydrogen peroxide in cells containing the Swedish APP mutation. This elevated activity is the result of the enhanced activation of both intrinsic and extrinsic apoptosis pathways, including activation of caspase 2 and caspase 8. Furthermore, we observed an enhanced activation of JNK pathway and an attenuation of apoptosis by SP600125, a JNK inhibitor, through protection of mitochondrial dysfunction and reduction of caspase 9 activity. Our findings provide evidence that the massive neurodegeneration in early age of FAD patients could be a result of an increased vulnerability of neurons through activation of different apoptotic pathways as a consequence of elevated levels of oxidative stress.

  10. The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species

    PubMed Central

    Ruefli, Astrid A.; Ausserlechner, Michael J.; Bernhard, David; Sutton, Vivien R.; Tainton, Kellie M.; Kofler, Reinhard; Smyth, Mark J.; Johnstone, Ricky W.

    2001-01-01

    Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death. PMID:11535817

  11. Pharmacological inhibition of caspase-8 limits lung tumour outgrowth

    PubMed Central

    Terlizzi, Michela; Di Crescenzo, Vincenzo Giuseppe; Perillo, Giuseppe; Galderisi, Antonio; Pinto, Aldo; Sorrentino, Rosalinda

    2015-01-01

    Background and Purpose Lung cancer is one of the leading causes of cancer death worldwide. Despite advances in therapy, conventional therapy is still the main treatment and has a high risk of chemotherapy resistance. Caspase-8 is involved in cell death and is a recognized marker for poor patient prognosis. Experimental Approach To elucidate the role of caspase-8 in lung carcinoma, we used human samples of non-small cell lung cancer (NSCLC) and a mouse model of carcinogen-induced lung cancer. Key Results Healthy and cancerous NSCLC samples had similar levels of the active form of caspase-8. Similarly, lung tumour-bearing mice had high levels of the active form of caspase-8. Pharmacological inhibition of caspase-8 by z-IETD-FMK robustly reduced tumour outgrowth and this was closely associated with a reduction in the release of pro-inflammatory cytokines, IL-6, TNF-α, IL-18, IL-1α, IL-33, but not IL-1β. Furthermore, inhibition of caspase-8 reduced the recruitment of innate suppressive cells, such as myeloid-derived suppressor cells, but not of regulatory T cells to lungs of tumour-bearing mice. However, despite the well-known role of caspase-8 in cell death, the apoptotic cascade (caspase-3, caspase-9 and Bcl-2 dependent) was not active in lungs of z-IETD-treated tumour-bearing mice, but instead higher levels of the short segment of c-FLIP (c-FLIPs) were detected. Similarly, human healthy lung samples had higher levels of c-FLIPs than cancerous samples. Conclusions and Implications Our data suggest that caspase-8 is an important orchestrator of cancer-associated inflammation and the presence of short segment of c-FLIP determines whether caspase-8 induces tumour proliferation or tumour arrest/regression in the lung. PMID:25917370

  12. Cleavage of cytoplasm within the oligonucleate zoosporangia of allomyces macrogynus.

    PubMed

    Ji, Yunjeong; Song, Youngsun; Kim, Namhun; Youn, Hyunjoo; Kang, Minkook; Song, Yurim; Cho, Chungwon

    2014-01-01

    Allomyces macrogynus produces zoosporangia that discharge uninucleate zoospores after cleavage of multinucleate cytoplasm. Cleavage of cytoplasm within the oligonucleate zoosporangia of A. macrogynus was visualized by constructing three-dimensional models based on electron micrographs and confocal images. In oligonucleate zoosporangia, three adjacent nuclei can form three cleavage planes with a line of intersection of the planes. The position and boundary of the cleavage planes are thought to be determined by the relative positions of the nuclei. The establishment of three cleavage planes by cleavage membranes occurred sequentially, and the nuclear axis connecting the centers of two nuclei affected the development of cleavage membranes on each cleavage plane. In multinucleate zoosporangia, groups of three neighboring nuclei near the cell cortex may initiate the sequential establishment of cleavage planes and then may interact with the nuclei further from the cortex until the interactions of nuclei are propagated to the central region of the cytoplasm. © 2014 by The Mycological Society of America.

  13. During Infection, Theiler's Virions Are Cleaved by Caspases and Disassembled into Pentamers

    PubMed Central

    Arslan, Sevim Yildiz; Son, Kyung-No

    2016-01-01

    ABSTRACT Infected macrophages in spinal cords of mice persistently infected with Theiler's murine encephalomyelitis virus (TMEV) undergo apoptosis, resulting in restricted virus yields, as do infected macrophages in culture. Apoptosis of murine macrophages in culture occurs via the intrinsic pathway later in infection (>10 h postinfection [p.i.]) after maximal virus titers (150 to 200 PFU/cell) have been reached, with loss of most infectious virus (<5 PFU/cell) by 20 to 24 h p.i. Here, we show that BeAn virus RNA replication, translation, polyprotein processing into final protein products, and assembly of protomers and pentamers in infected M1-D macrophages did not differ from those processes in TMEV-infected BHK-21 cells, which undergo necroptosis. However, the initial difference from BHK-21 cell infection was seen at 10 to 12 h p.i., where virions from the 160S peak in sucrose gradients had incompletely processed VP0 (compared to that in infected BHK-21 cells). Thereafter, there was a gradual loss of the 160S virion peak in sucrose gradients, with replacement by a 216S peak that was observed to contain pentamers among lipid debris in negatively stained grids by electron microscopy. After infection or incubation of purified virions with activated caspase-3 in vitro, 13- and 17-kDa capsid peptide fragments were observed and were predicted by algorithms to contain cleavage sites within proteins by cysteine-dependent aspartate-directed proteases. These findings suggest that caspase cleavage of sites in exposed capsid loops of assembled virions occurs contemporaneously with the onset and progression of apoptosis later in the infection. IMPORTANCE Theiler's murine encephalomyelitis virus (TMEV) infection in mice results in establishment of virus persistence in the central nervous system and chronic inflammatory demyelinating disease, providing an experimental animal model for multiple sclerosis. Virus persistence takes place primarily in macrophages recruited into the

  14. Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

    PubMed Central

    Schoberle, Taylor J.; Chung, Lawton K.; McPhee, Joseph B.; Bogin, Ben

    2016-01-01

    Pathogenic Yersinia species utilize a type III secretion system to translocate Yop effectors into infected host cells. Yop effectors inhibit innate immune responses in infected macrophages to promote Yersinia pathogenesis. In turn, Yersinia-infected macrophages respond to translocation of Yops by activating caspase-1, but different mechanisms of caspase-1 activation occur, depending on the bacterial genotype and the state of phagocyte activation. In macrophages activated with lipopolysaccharide (LPS) prior to Yersinia pseudotuberculosis infection, caspase-1 is activated by a rapid inflammasome-dependent mechanism that is inhibited by translocated YopM. The possibility that other effectors cooperate with YopM to inhibit caspase-1 activation in LPS-activated macrophages has not been investigated. Toward this aim, epistasis analysis was carried out in which the phenotype of a Y. pseudotuberculosis yopM mutant was compared to that of a yopJ yopM, yopE yopM, yopH yopM, yopT yopM, or ypkA yopM mutant. Activation of caspase-1 was measured by cleavage of the enzyme, release of interleukin-1β (IL-1β), and pyroptosis in LPS-activated macrophages infected with wild-type or mutant Y. pseudotuberculosis strains. Results show enhanced activation of caspase-1 after infection with the yopJ yopM mutant relative to infection by any other single or double mutant. Similar results were obtained with the yopJ, yopM, and yopJ yopM mutants of Yersinia pestis. Following intravenous infection of mice, the Y. pseudotuberculosis yopJ mutant was as virulent as the wild type, while the yopJ yopM mutant was significantly more attenuated than the yopM mutant. In summary, through epistasis analysis this work uncovered an important role for YopJ in inhibiting caspase-1 in activated macrophages and in promoting Yersinia virulence. PMID:26810037

  15. ROS and ERK1/2-mediated caspase-9 activation increases XAF1 expression in dexamethasone-induced apoptosis of EBV-transformed B cells.

    PubMed

    Park, Ga Bin; Choi, Yunock; Kim, Yeong Seok; Lee, Hyun-Kyung; Kim, Daejin; Hur, Dae Young

    2013-07-01

    Dexamethasone (Dex) inhibits the growth of diverse types of cancer cells and is utilized clinically for the therapy of hematological malignancies. In this study, we investigated the molecular mechanisms of Dex action in the apoptosis of Epstein-Barr virus (EBV)-transformed B cells. We showed that Dex inhibited the proliferation of EBV-transformed B cells and induced apoptosis by activating caspase-9, -3 and -8. While activation of caspase-9 was triggered as early as 2 h after Dex treatment, cleavage of caspase-8 was deferred and was found 8 h after the exposure. Dex-dependent activation of caspase-8 was blocked by the specific caspase-9 inhibitor, z-LEHD-fmk. Moreover, Dex significantly increased the expression of X-linked inhibitor of apoptosis (XIAP)‑associated factor 1 (XAF1) and induced the translocation of XAF1 into the cytosol. Cytosolic XAF1 with Puma induced the translocation of Bax into mitochondria. Dex led to up-regulation of reactive oxygen species (ROS) generation and the phosphorylation of ERK1/2 after the exposure. We speculated that ROS generation might be the first event of Dex-induced apoptosis because ROS inhibitor NAC abrogated ROS production and ERK1/2 activation, but PD98059 did not block ROS production. NAC and PD98059 also suppressed the translocation of XAF1, Puma and Bax into mitochondria. These results demonstrated that Dex-mediated activation of caspase-9 via ROS generation and ERK1/2 pathway activation resulted in the activation of caspase-8 and the increment of XAF1, thereby induced apoptosis of EBV-transformed B cells. These findings suggest that Dex constitutes a probable therapy for EBV-associated hematological malignancies.

  16. Proteinase 3 and Serpin B1: a novel pathway in the regulation of caspase-3 activation, neutrophil spontaneous apoptosis, and inflammation.

    PubMed

    Loison, Fabien; Xu, Yuanfu; Luo, Hongbo R

    Neutrophils are the first responders of the inflammatory response. They are characterized by their potent cytotoxic content but also by their limited lifetime. This short half-life is thought to be a self-protecting mechanism for the host, as highlighted by the numerous pathologies associated with imbalanced neutrophil survival. Neutrophil spontaneous death is the prototype of programmed cell death, harboring all the phenotypic hallmarks of apoptosis and dependent on the activation of the effector caspase-3. However, the pathways regulating neutrophil spontaneous death remain ill-defined. In a recent publication, we determined that in aging neutrophils, the cleavage and activation of caspase-3 was mediated by the serine protease Proteinase 3 (PR3), and was independent of the canonical extrinsic and intrinsic apoptosis pathways. In mature neutrophils, PR3 was stored in granules and progressively released to the cytosol during neutrophil aging. The release of PR3 was dependent on lysosomal membrane permeabilization (LMP). Once in the cytosol, PR3 cleaved procaspase-3 at a site upstream of the caspase-9 cleavage site, leading to caspase-3 activation. Inhibition, knockdown or knockout of PR3 delayed neutrophil apoptosis in vitro and in vivo. The adoptive transfer of both WT and PR3-deficient neutrophils to WT mice revealed that the delayed death of neutrophils lacking PR3 in vivo was due to an altered intrinsic apoptosis/survival pathway and not to difference in the inflammatory microenvironment. The cytosolic inhibitor of serine proteases serpin b1 counterbalanced the activity of PR3 in the cytosol of neutrophils, and the deletion of serpinb1 in neutrophils accelerated their spontaneous death. In summary, our results reveal that PR3 and serpinB1 are part of a newly characterized apoptosis pathway, regulating caspase-3 activation and neutrophil spontaneous death and the survival of neutrophils during inflammation.

  17. Proteinase 3 and Serpin B1: a novel pathway in the regulation of caspase-3 activation, neutrophil spontaneous apoptosis, and inflammation

    PubMed Central

    Xu, Yuanfu; Luo, Hongbo R

    2015-01-01

    Neutrophils are the first responders of the inflammatory response. They are characterized by their potent cytotoxic content but also by their limited lifetime. This short half-life is thought to be a self-protecting mechanism for the host, as highlighted by the numerous pathologies associated with imbalanced neutrophil survival. Neutrophil spontaneous death is the prototype of programmed cell death, harboring all the phenotypic hallmarks of apoptosis and dependent on the activation of the effector caspase-3. However, the pathways regulating neutrophil spontaneous death remain ill-defined. In a recent publication, we determined that in aging neutrophils, the cleavage and activation of caspase-3 was mediated by the serine protease Proteinase 3 (PR3), and was independent of the canonical extrinsic and intrinsic apoptosis pathways. In mature neutrophils, PR3 was stored in granules and progressively released to the cytosol during neutrophil aging. The release of PR3 was dependent on lysosomal membrane permeabilization (LMP). Once in the cytosol, PR3 cleaved procaspase-3 at a site upstream of the caspase-9 cleavage site, leading to caspase-3 activation. Inhibition, knockdown or knockout of PR3 delayed neutrophil apoptosis in vitro and in vivo. The adoptive transfer of both WT and PR3-deficient neutrophils to WT mice revealed that the delayed death of neutrophils lacking PR3 in vivo was due to an altered intrinsic apoptosis/survival pathway and not to difference in the inflammatory microenvironment. The cytosolic inhibitor of serine proteases serpin b1 counterbalanced the activity of PR3 in the cytosol of neutrophils, and the deletion of serpinb1 in neutrophils accelerated their spontaneous death. In summary, our results reveal that PR3 and serpinB1 are part of a newly characterized apoptosis pathway, regulating caspase-3 activation and neutrophil spontaneous death and the survival of neutrophils during inflammation. PMID:26029732

  18. Transgenic mice expressing caspase-6-derived N-terminal fragments of mutant huntingtin develop neurologic abnormalities with predominant cytoplasmic inclusion pathology composed largely of a smaller proteolytic derivative

    PubMed Central

    Tebbenkamp, Andrew T.N.; Green, Cameron; Xu, Guilian; Denovan-Wright, Eileen M.; Rising, Aaron C.; Fromholt, Susan E.; Brown, Hilda H.; Swing, Debbie; Mandel, Ronald J.; Tessarollo, Lino; Borchelt, David R.

    2011-01-01

    Recent studies have implicated an N-terminal caspase-6 cleavage product of mutant huntingtin (htt) as an important mediator of toxicity in Huntington's disease (HD). To directly assess the consequences of such fragments on neurologic function, we produced transgenic mice that express a caspase-6 length N-terminal fragment of mutant htt (N586) with both normal (23Q) and disease (82Q) length glutamine repeats. In contrast to mice expressing N586-23Q, mice expressing N586-82Q accumulate large cytoplasmic inclusion bodies that can be visualized with antibodies to epitopes throughout the N586 protein. However, biochemical analyses of aggregated mutant huntingtin in these mice demonstrated that the inclusion bodies are composed largely of a much smaller htt fragment (terminating before residue 115), with lesser amounts of full-length N586-82Q fragments. Mice expressing the N586-82Q fragment show symptoms typical of previously generated mice expressing mutant huntingtin fragments, including failure to maintain weight, small brain weight and reductions in specific mRNAs in the striatum. Uniquely, these N586-82Q mice develop a progressive movement disorder that includes dramatic deficits in motor performance on the rotarod and ataxia. Our findings suggest that caspase-6-derived fragments of mutant htt are capable of inducing novel HD-related phenotypes, but these fragments are not terminal cleavage products as they are subject to further proteolysis. In this scenario, mutant htt fragments derived from caspase 6, or possibly other proteases, could mediate HD pathogenesis via a ‘hit and run' type of mechanism in which caspase-6, or other larger N-terminal fragments, mediate a neurotoxic process before being cleaved to a smaller fragment that accumulates pathologically. PMID:21515588

  19. Granzyme B cleavage of autoantigens in autoimmunity

    PubMed Central

    Darrah, Erika; Rosen, Antony

    2011-01-01

    The systemic autoimmune diseases are a complex group of disorders characterized by elaboration of high titer autoantibodies and immune-mediated damage of tissues. Two striking features of autoimmune rheumatic diseases are their self-sustaining nature and capacity for auto-amplification, exemplified by disease flares. These features suggest the presence of a feed-forward cycle in disease propagation, in which immune effector pathways drive the generation/release of autoantigens, which in turn fuel the immune response. There is a growing awareness that structural modification during cytotoxic granule-induced cell death is a frequent and striking feature of autoantigens, and may be an important principle driving disease. This review focuses on granzyme B (GrB)-mediated cleavage of autoantigens including (i) features of GrB cleavage sites within autoantigens, (ii) co-location of cleavage sites with autoimmune epitopes, and (iii) GrB-sensitivity of autoantigens in disease-relevant target tissue. The mechanisms whereby GrB-induced changes in autoantigen structure may contribute to the initiation and propagation of autoimmunity are reviewed and reveal that GrB has the potential to create or destroy autoimmune epitopes. As there remains no direct evidence demonstrating a causal role for GrB-cleavage of antigens in the generation of autoimmunity, this review highlights important outstanding questions about the role of GrB in autoantigen selection. PMID:20075942

  20. Turning on caspases with genetics and small molecules.

    PubMed

    Morgan, Charles W; Julien, Olivier; Unger, Elizabeth K; Shah, Nirao M; Wells, James A

    2014-01-01

    Caspases, aspartate-specific cysteine proteases, have fate-determining roles in many cellular processes including apoptosis, differentiation, neuronal remodeling, and inflammation (for review, see Yuan & Kroemer, 2010). There are a dozen caspases in humans alone, yet their individual contributions toward these phenotypes are not well understood. Thus, there has been considerable interest in activating individual caspases or using their activity to drive these processes in cells and animals. We envision that such experimental control of caspase activity can not only afford novel insights into fundamental biological problems but may also enable new models for disease and suggest possible routes to therapeutic intervention. In particular, localized, genetic, and small-molecule-controlled caspase activation has the potential to target the desired cell type in a tissue. Suppression of caspase activation is one of the hallmarks of cancer and thus there has been significant enthusiasm for generating selective small-molecule activators that could bypass upstream mutational events that prevent apoptosis. Here, we provide a practical guide that investigators have devised, using genetics or small molecules, to activate specific caspases in cells or animals. Additionally, we show genetically controlled activation of an executioner caspase to target the function of a defined group of neurons in the adult mammalian brain.

  1. Pyroptosis: Caspase-11 Unlocks the Gates of Death.

    PubMed

    de Gassart, Aude; Martinon, Fabio

    2015-11-17

    How inflammatory caspases trigger pyroptotic cell death is mostly unexplained. In this issue of Immunity, Núñez and colleagues report that caspase-11 cleaves the transmembrane channel pannexin-1, causing an efflux of cellular ATP that promotes a P2X7 receptor-dependent pyroptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Differential expression of Livin, caspase-3, and second mitochondria-derived activator of caspases in chronic rhinosinusitis with nasal polyps.

    PubMed

    Lin, Hai; Lin, Dong; Xiong, Xisheng

    2014-12-01

    The pathogenesis of human chronic rhinosinusitis with nasal polyps (CRSwNP) remains undetermined. Livin is a member of the inhibitor of the apoptosis protein family proteins. Caspase-3 and second mitochondria-derived activator of caspases (Smac) are critical in the induction of apoptosis. However, little is known about their roles in CRSwNP. We aimed to investigate the expression and role of Livin, caspase-3, and Smac in CRSwNP. Basic research and descriptive study. Fuzhou General Hospital, Fuzhou, Fujian, China. The immunohistochemistry method was employed for detecting Livin, caspase-3, and Smac protein expression, and real-time polymerase chain reaction was used for assaying mRNA expression of Livin, caspase-3, and Smac in CRSwNP and controls. Moreover, the effects of various stimulators on Livin were evaluated on human nasal epithelial cells (HNECs) culture. Then, the effects of Livin on caspase-3 and Smac were observed on the culture of HNECs. Stronger protein and mRNA expression of Livin was observed in CRSwNP, especially eosinophilic CRSwNP, weaker protein and mRNA expression of caspase-3 and Smac was observed in CRSwNP, and Livin expression was negatively related to caspase-3 or Smac expression, respectively. Livin mRNA was augmented by interleukin (IL)-4, IL-17A, and IL-1β but suppressed by interferon-γ. Caspase-3 and Smac mRNA expression were inhibited by Livin. Upregulation of Livin and downregulation of caspase-3 and Smac were observed in CRSwNP, especially in eosinophilic CRSwNP. Livin may exert its anti-apoptosis effect by suppressing caspase-3 and Smac in CRSwNP. IL-4, IL-17A, and IL-1β may be critical for Livin expression. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014.

  3. Mechanisms of cancer chemoprevention by hop bitter acids (beer aroma) through induction of apoptosis mediated by Fas and caspase cascades.

    PubMed

    Chen, Wei-Jen; Lin, Jen-Kun

    2004-01-14

    The bitter acids of hops (Humulus lupulus L.) mainly consist of alpha-acids, beta-acids, and their oxidation products that contribute the unique aroma of the beer beverage. Hop bitter acids displayed a strong growth inhibitory effect against human leukemia HL-60 cells, with an estimated IC(50) value of 8.67 microg/mL, but were less effective against human histolytic lymphoma U937 cells. Induction of apoptosis was confirmed in HL-60 cells by DNA fragmentation and the appearance of a sub-G1 DNA peak, which were preceded by dissipation of mitochondrial membrane potential, cytochrome c release, and subsequent induction of pro-caspase-9 and -3 processing. Cleavages of PARP and DFF-45 were accompanied with activation of caspase-9 and -3 triggered by hop bitter acids in HL-60 cells. The change in the expression of Bcl-2, Bcl-X(L), and Bax in response to hop bitter acids was studied, and the Bcl-2 protein level slightly decreased; however, the Bcl-X(L) protein level was obviously decreased, whereas the Bax protein level was dramatically increased, indicating that the control of Bcl-2 family proteins by hop bitter acids might participate in the disruption of mitochondrial integrity. In addition, the results showed that hop bitter acids promoted the up-regulation of Fas and FasL prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in hop bitter acids-induced cells. Taken together, these findings suggest that a certain intimate link might exist between receptor- and mitochondria-mediated death signalings that committed to cell death induced by hop bitter acids. The induction of apoptosis by hop bitter acids may offer a pivotal mechanism for their chemopreventive action.

  4. A caspase-related protease regulates apoptosis in yeast.

    PubMed

    Madeo, Frank; Herker, Eva; Maldener, Corinna; Wissing, Silke; Lächelt, Stephan; Herlan, Mark; Fehr, Markus; Lauber, Kirsten; Sigrist, Stephan J; Wesselborg, Sebastian; Fröhlich, Kai Uwe

    2002-04-01

    Yeast can undergo cell death accompanied by cellular markers of apoptosis. However, orthologs of classical mammalian apoptosis regulators appeared to be missing from the yeast genome, challenging a common mechanism of yeast and mammalian apoptosis. Here we investigate Yor197w, a yeast protein with structural homology to mammalian caspases, and demonstrate caspase-like processing of the protein. Hydrogen peroxide treatment induces apoptosis together with a caspase-like enzymatic activity in yeast. This response is completely abrogated after disruption and strongly stimulated after overexpression of Yor197w. Yor197w also mediates the death process within chronologically aged cultures, pointing to a physiological role in elimination of overaged cells. We conclude that Yor197w indeed functions as a bona fide caspase in yeast and propose the name Yeast Caspase-1 (YCA1, gene YCA1).

  5. Suppression of caspase-11 expression by histone deacetylase inhibitors

    SciTech Connect

    Heo, Hyejung; Yoo, Lang; Shin, Ki Soon; Kang, Shin Jung

    2009-01-02

    It has been well documented that histone deacetylase inhibitors suppress inflammatory gene expression. Therefore, we investigated whether histone deacetylase inhibitors modulate the expression of caspase-11 that is known as an inducible caspase regulating both inflammation and apoptosis. In the present study, we show that sodium butyrate and trichostatin A, two structurally unrelated inhibitors of histone deacetylase (HDAC), effectively suppressed the induction of caspase-11 in mouse embryonic fibroblasts stimulated with lipopolysaccharides. Sodium butyrate inhibited the activation of upstream signaling events for the caspase-11 induction such as activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, degradation of inhibitor of {kappa}B, and activation of nuclear factor-{kappa}B. These results suggest that the HDAC inhibitor suppressed cytosolic signaling events for the induction of caspase-11 by inhibiting the deacetylation of non-histone proteins.

  6. Apoptosis in Heart Failure: Release of Cytochrome c from Mitochondria and Activation of Caspase-3 in Human Cardiomyopathy

    NASA Astrophysics Data System (ADS)

    Narula, Jagat; Pandey, Pramod; Arbustini, Eloisa; Haider, Nezam; Narula, Navneet; Kolodgie, Frank D.; dal Bello, Barbara; Semigran, Marc J.; Bielsa-Masdeu, Anna; Dec, G. William; Israels, Sara; Ballester, Manel; Virmani, Renu; Saxena, Satya; Kharbanda, Surender

    1999-07-01

    Apoptosis has been shown to contribute to loss of cardiomyocytes in cardiomyopathy, progressive decline in left ventricular function, and congestive heart failure. Because the molecular mechanisms involved in apoptosis of cardiocytes are not completely understood, we studied the biochemical and ultrastructural characteristics of upstream regulators of apoptosis in hearts explanted from patients undergoing transplantation. Sixteen explanted hearts from patients undergoing heart transplantation were studied by electron microscopy or immunoblotting to detect release of mitochondrial cytochrome c and activation of caspase-3. The hearts explanted from five victims of motor vehicle accidents or myocardial ventricular tissues from three donor hearts were used as controls. Evidence of apoptosis was observed only in endstage cardiomyopathy. There was significant accumulation of cytochrome c in the cytosol, over myofibrils, and near intercalated discs of cardiomyocytes in failing hearts. The release of mitochondrial cytochrome c was associated with activation of caspase-3 and cleavage of its substrate protein kinase C δ but not poly(ADP-ribose) polymerase. By contrast, there was no apparent accumulation of cytosolic cytochrome c or caspase-3 activation in the hearts used as controls. The present study provides in vivo evidence of cytochrome c-dependent activation of cysteine proteases in human cardiomyopathy. Activation of proteases supports the phenomenon of apoptosis in myopathic process. Because loss of myocytes contributes to myocardial dysfunction and is a predictor of adverse outcomes in the patients with congestive heart failure, the present demonstration of an activated apoptotic cascade in cardiomyopathy could provide the basis for novel interventional strategies.

  7. NPM-RAR binding to TRADD selectively inhibits caspase activation, while allowing activation of NFκB and JNK.

    PubMed

    Chattopadhyay, Anuja; Abecassis, Irina; Redner, Robert L

    2015-01-01

    The t(5;17) variant of acute promeylocytic leukemia (APL) expresses a fusion of nucleophosmin (NPM) with the retinoic acid receptor alpha (RARA). We have previously shown that NPM-RAR is a binding partner of the tumor necrosis factor (TNF) receptor type-I-associated DEATH domain protein, TRADD. Binding of TNF to its receptor, TNF-R, induces recruitment of TRADD, and subsequent recruitment of a cascade of proteins that ultimate activate caspase 3, nuclear factor κB (NFκB) and c-Jun N-terminal kinase (JNK). We have previously shown that NPM-RAR interaction with TRADD blocks TNF activation of caspase 3, caspase 8, poly(ADP-ribose) polymerase (PARP) cleavage and, ultimately, apoptosis. We now report that NPM-RAR expression is permissive for TNF activation of NFκB and JNK. We propose that inhibition of TNF activation of apoptosis, while preserving TNF activation of NFκB and JNK pathways that stimulate cell growth and survival, represents a novel mechanism through which NPM-RAR contributes to development of the leukemic phenotype.

  8. Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation

    PubMed Central

    Paulsson, Johan F.; Schultz, Sebastian W.; Köhler, Martin; Leibiger, Ingo; Berggren, Per-Olof; Westermark, Gunilla T.

    2008-01-01

    Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP) . Peptides were added in solubilized from (50 μM) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occure in cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes. PMID:18566681

  9. Mycobacterium tuberculosis PPE32 promotes cytokines production and host cell apoptosis through caspase cascade accompanying with enhanced ER stress response

    PubMed Central

    Zeng, Jie; Abdalla, Abualgasim Elgaili; Xie, Jianping

    2016-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis (MTB) infection, remains a grave global public health burden which claims the lives around two to three million annually. PE and PPE proteins, featured by the Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs at the conserved N-terminal domain, are abundant in the MTB genome. PPE32 can increase intracellular survival of mycobacteria through abnormally increase in cytokines production. PPE32 might subvert the macrophage immune response and thwart its bactericidal effect. THP-1 macrophages treated with PPE32 or infected with Mycobacterium smegmatis (MS) expression PPE32 showed increase of cytokines production and multiple hallmarks of apoptosis. We found that PPE32 significantly increases the expression of IL-12p40 and IL-32 through ERK1/2 signaling pathway. In addition, the cell viability of macrophage was inhibited after PPE32 stimulation. We noted that PPE32 induces cleavage of caspase-3 and caspase-9, while inhibition of caspase activity significantly abrogates the PPE32-induced cell apoptosis. Moreover, PPE32 treatment promotes endoplasmic reticulum stress related gene expression, suggesting ER stress might be responsible for PPE32-induced cell apoptosis. PMID:27634911

  10. p53 facilitates pRb cleavage in IL-3-deprived cells: novel pro-apoptotic activity of p53.

    PubMed Central

    Gottlieb, E; Oren, M

    1998-01-01

    In the interleukin-3 (IL-3)-dependent lymphoid cell line DA-1, functional p53 is required for efficient apoptosis in response to IL-3 withdrawal. Activation of p53 in these cells, by either DNA damage or p53 overexpression, results in a vital growth arrest in the presence of IL-3 and in accelerated apoptosis in its absence. Thus, IL-3 can control the choice between p53-dependent cell-cycle arrest and apoptosis. Here we report that the cross-talk between p53 and IL-3 involves joint control of pRb cleavage and degradation. Depletion of IL-3 results in caspase-mediated pRb cleavage, occurring preferentially within cells which express functional p53. Moreover, pRb can be cleaved efficiently by extracts prepared from DA-1 cells but not from their derivatives which lack p53 function. Inactivation of pRb through expression of the human papillomavirus (HPV) E7 oncogene overrides the effect of IL-3 in a p53-dependent manner. Our data suggest a novel role for p53 in the regulation of cell death and a novel mechanism for the cooperation between p53 and survival factor deprivation. Thus, p53 makes cells permissive to pRb cleavage, probably by controlling the potential activity of a pRb-cleaving caspase, whereas IL-3 withdrawal provides signals that turn on this potential activity and lead to the actual cleavage and subsequent degradation of pRb. Elimination of a presumptive anti-apoptotic effect of pRb may then facilitate conversion of p53-mediated growth arrest into apoptosis. PMID:9649429

  11. Grape seed extract induces apoptotic death of human prostate carcinoma DU145 cells via caspases activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release.

    PubMed

    Agarwal, Chapla; Singh, Rana P; Agarwal, Rajesh

    2002-11-01

    Grape seed extract (GSE), rich in the bioflavonoids commonly known as procyanidins, is one of the most commonly consumed dietary supplements in the United States because of its several health benefits. Epidemiological studies show that many prostate cancer (PCA) patients use herbal extracts as dietary supplements in addition to their prescription drugs. Accordingly, in recent years, we have focused our attention on assessing the efficacy of GSE against PCA. Our studies showed that GSE inhibits growth and induces apoptotic death of human PCA cells in culture and in nude mice. Here, we performed detailed studies to define the molecular mechanism of GSE-induced apoptosis in advanced human PCA DU145 cells. GSE treatment of cells at various doses (50-200 micro g/ml) for 12-72 h resulted in a moderate to strong apoptotic death in a dose- and time-dependent manner. In the studies assessing the apoptotic-signaling pathway induced by GSE, we observed an increase in cleaved fragments of caspases 3, 7 and 9 as well as PARP in GSE-treated cells after 48 and 72 h of treatment. Pre-treatment of cells with general caspases inhibitor, z-Val-Ala-Asp(OMe)-FMK or caspase 3-like proteases inhibitor [z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK], almost completely (approximately 90%) inhibited the GSE-induced apoptotic cell death. In a later case, GSE-induced caspase-3 activity was completely inhibited. Selective caspase 9 inhibitor [z-Leu-Glu(OMe)-His-Asp(OMe)-FMK] showed only partial inhibition of GSE-induced apoptosis whereas GSE-induced protease activity of caspase 9 was completely inhibited. Upstream of caspase cascade, GSE showed disappearance of mitochondrial membrane potential and an increase in cytochrome c release in cytosol. Together, these results suggest that GSE possibly causes mitochondrial damage leading to cytochrome c release in cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic death of human PCA DU145 cells. Furthermore, GSE

  12. 5-Fluorouracil-induced apoptosis in colorectal cancer cells is caspase-9-dependent and mediated by activation of protein kinase C-δ

    PubMed Central

    MHAIDAT, NIZAR M.; BOUKLIHACENE, MOHAMMED; THORNE, RICK F.

    2014-01-01

    Elucidation of the molecular mechanisms by which 5-fluorouracil (5-FU) induces apoptosis is required in order to understand the resistance of colorectal cancer (CRC) cells to 5-FU. In the current study, 5-FU-induced apoptosis was assessed using the propidium iodide method. Involvement of protein kinase C (PKC) was assessed by evaluating the extent of their activation in CRC, following treatment with 5-FU, using biochemical inhibitors and western blot analysis. The results revealed that 5-FU induces varying degrees of apoptosis in CRC cells; HCT116 cells were identified to be the most sensitive cells and SW480 were the least sensitive. In addition, 5-FU-induced apoptosis was caspase-dependent as it appeared to be initiated by caspase-9. Furthermore, PKCɛ was marginally expressed in CRC cells and no changes were observed in the levels of cleavage or phosphorylation following treatment with 5-FU. The treatment of HCT116 cells with 5-FU increased the expression, phosphorylation and cleavage of PKCδ. The inhibition of PKCδ was found to significantly inhibit 5-FU-induced apoptosis. These results indicated that 5-FU induces apoptosis in CRC by the activation of PKCδ and caspase-9. In addition, the levels of PKCδ activation may determine the sensitivity of CRC to 5-FU. PMID:25013487

  13. Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag.

    PubMed

    Shahravan, S Hesam; Qu, Xuanlu; Chan, I-San; Shin, Jumi A

    2008-06-01

    In our work with designed minimalist proteins based on the bZIP motif, we have found our His-tagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6-C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1-4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more "open" protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of related sequence.

  14. Molecular Dynamics Studies of Caspase-3

    PubMed Central

    Sulpizi, M.; Rothlisberger, U.; Carloni, P.

    2003-01-01

    Caspase-3 is a fundamental target for pharmaceutical interventions against a variety of diseases involving disregulated apoptosis. The enzyme is active as a dimer with two symmetry-related active sites, each featuring a Cys-His catalytic dyad and a selectivity loop, which recognizes the characteristic DEVD pattern of the substrate. Here, a molecular dynamics study of the enzyme in complex with two pentapeptide substrates DEVDG is presented, which provides a characterization of the dynamic properties of the active form in aqueous solution. The mobility of the substrate and that of the catalytic residues are rather low indicating a distinct preorganization effect of the Michaelis complex. An essential mode analysis permits us to identify coupled motions between the two monomers. In particular, it is found that the motions of the two active site loops are correlated and tend to steer the substrate toward the reactive center, suggesting that dimerization has a distinct effect on the dynamic properties of the active site regions. The selectivity loop of one monomer turns out to be correlated with the N-terminal region of the p12 subunit of the other monomer, an interaction that is also found to play a fundamental role in the electrostatic stabilization of the quaternary structure. To further characterize the specific influence of dimerization on the enzyme essential motions, a molecular dynamics analysis is also performed on the isolated monomer. PMID:12668429

  15. Zinc-mediated Allosteric Inhibition of Caspase-6*

    PubMed Central

    Velázquez-Delgado, Elih M.; Hardy, Jeanne A.

    2012-01-01

    Zinc and caspase-6 have independently been implicated in several neurodegenerative disorders. Depletion of zinc intracellularly leads to apoptosis by an unknown mechanism. Zinc inhibits cysteine proteases, including the apoptotic caspases, leading to the hypothesis that zinc-mediated inhibition of caspase-6 might contribute to its regulation in a neurodegenerative context. Using inductively coupled plasma optical emission spectroscopy, we observed that caspase-6 binds one zinc per monomer, under the same conditions where the zinc leads to complete loss of enzymatic activity. To understand the molecular details of zinc binding and inhibition, we performed an anomalous diffraction experiment above the zinc edge. The anomalous difference maps showed strong 5σ peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site. Zinc was not observed bound to the active site. The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family. Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation. PMID:22891250

  16. Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy

    PubMed Central

    Putinski, Charis; Abdul-Ghani, Mohammad; Stiles, Rebecca; Brunette, Steve; Dick, Sarah A.; Fernando, Pasan; Megeney, Lynn A.

    2013-01-01

    Cardiomyocyte hypertrophy is the cellular response that mediates pathologic enlargement of the heart. This maladaptation is also characterized by cell behaviors that are typically associated with apoptosis, including cytoskeletal reorganization and disassembly, altered nuclear morphology, and enhanced protein synthesis/translation. Here, we investigated the requirement of apoptotic caspase pathways in mediating cardiomyocyte hypertrophy. Cardiomyocytes treated with hypertrophy agonists displayed rapid and transient activation of the intrinsic-mediated cell death pathway, characterized by elevated levels of caspase 9, followed by caspase 3 protease activity. Disruption of the intrinsic cell death pathway at multiple junctures led to a significant inhibition of cardiomyocyte hypertrophy during agonist stimulation, with a corresponding reduction in the expression of known hypertrophic markers (atrial natriuretic peptide) and transcription factor activity [myocyte enhancer factor-2, nuclear factor kappa B (NF-κB)]. Similarly, in vivo attenuation of caspase activity via adenoviral expression of the biologic effector caspase inhibitor p35 blunted cardiomyocyte hypertrophy in response to agonist stimulation. Treatment of cardiomyocytes with procaspase 3 activating compound 1, a small-molecule activator of caspase 3, resulted in a robust induction of the hypertrophy response in the absence of any agonist stimulation. These results suggest that caspase-dependent signaling is necessary and sufficient to promote cardiomyocyte hypertrophy. These results also confirm that cell death signal pathways behave as active remodeling agents in cardiomyocytes, independent of inducing an apoptosis response. PMID:24101493

  17. Promoting elongation with transcript cleavage stimulatory factors.

    PubMed

    Fish, Rachel N; Kane, Caroline M

    2002-09-13

    Transcript elongation by RNA polymerase is a dynamic process, capable of responding to a number of intrinsic and extrinsic signals. A number of elongation factors have been identified that enhance the rate or efficiency of transcription. One such class of factors facilitates RNA polymerase transcription through blocks to elongation by stimulating the polymerase to cleave the nascent RNA transcript within the elongation complex. These cleavage factors are represented by the Gre factors from prokaryotes, and TFIIS and TFIIS-like factors found in archaea and eukaryotes. High-resolution structures of RNA polymerases and the cleavage factors in conjunction with biochemical investigations and genetic analyses have provided insights into the mechanism of action of these elongation factors. However, there are yet many unanswered questions regarding the regulation of these factors and their effects on target genes.

  18. Unexpected Trypsin Cleavage at Ubiquitinated Lysines

    PubMed Central

    2015-01-01

    Unexpected tryptic cleavage has been characterized at modified K48 residues in polyubiquitins. In particular, the tryptic products of all seven of the lysine-linked dimers of ubiquitin and of three trimers—linear Ub–48Ub–48Ub, linear Ub–63Ub–63Ub, and the branched trimer [Ub]2–6,48Ub—have been analyzed. In addition to the peptide products expected under commonly used tryptic conditions, we observe that peptides are formed with an unexpected ε-glycinylglycinyl-Lys carboxyl terminus when the site of linkage is Lys48. Trypsin from three different commercial sources exhibited this aberration. Initial cleavage at R74 is proposed in a distal ubiquitin to produce a glycinylglycinyl-lysine residue which is bound by trypsin. PMID:26182167

  19. [Laparoscopic cleavage in splenic symptomatic cyst].

    PubMed

    Fernández-López, Antonio-José; Candel-Arenas, Marifé; González-Valverde, Francisco-Miguel; Luján-Martínez, Delia; Medina-Manuel, Esther; Albarracín Marín-Blázquez, Antonio

    2016-12-30

    Splenic cysts are rare diseases that are diagnosed incidentally during imaging studies. When cysts are recognized, surgical treatment is recommended adapted to the particular case, depending on the size and location of the cyst and the age of the patient in order to avoid dangerous complications such as spleen rupture or cyst infection with abscess. We report 2patients with symptomatic splenic epidermoid cyst treated by laparoscopic cleavage. Laparoscopic cleavage is a surgical option for splenic cyst, with the goal of reducing postoperative complications while preserving splenic function. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

  20. Preventing cleavage of Mer promotes efferocytosis and suppresses acute lung injury in bleomycin treated mice

    SciTech Connect

    Lee, Ye-Ji; Lee, Seung-Hae; Youn, Young-So; Choi, Ji-Yeon; Song, Keung-Sub; Cho, Min-Sun; Kang, Jihee Lee

    2012-08-15

    Mer receptor tyrosine kinase (Mer) regulates macrophage activation and promotes apoptotic cell clearance. Mer activation is regulated through proteolytic cleavage of the extracellular domain. To determine if membrane-bound Mer is cleaved during bleomycin-induced lung injury, and, if so, how preventing the cleavage of Mer enhances apoptotic cell uptake and down-regulates pulmonary immune responses. During bleomycin-induced acute lung injury in mice, membrane-bound Mer expression decreased, but production of soluble Mer and activity as well as expression of disintegrin and metalloproteinase 17 (ADAM17) were enhanced . Treatment with the ADAM inhibitor TAPI-0 restored Mer expression and diminished soluble Mer production. Furthermore, TAPI-0 increased Mer activation in alveolar macrophages and lung tissue resulting in enhanced apoptotic cell clearance in vivo and ex vivo by alveolar macrophages. Suppression of bleomycin-induced pro-inflammatory mediators, but enhancement of hepatocyte growth factor induction were seen after TAPI-0 treatment. Additional bleomycin-induced inflammatory responses reduced by TAPI-0 treatment included inflammatory cell recruitment into the lungs, levels of total protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, as well as caspase-3 and caspase-9 activity and alveolar epithelial cell apoptosis in lung tissue. Importantly, the effects of TAPI-0 on bleomycin-induced inflammation and apoptosis were reversed by coadministration of specific Mer-neutralizing antibodies. These findings suggest that restored membrane-bound Mer expression by TAPI-0 treatment may help resolve lung inflammation and apoptosis after bleomycin treatment. -- Highlights: ►Mer expression is restored by TAPI-0 treatment in bleomycin-stimulated lung. ►Mer signaling is enhanced by TAPI-0 treatment in bleomycin-stimulated lung. ►TAPI-0 enhances efferocytosis and promotes resolution of lung injury.

  1. The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program.

    PubMed

    Elliott, K; Ge, K; Du, W; Prendergast, G C

    2000-09-28

    Cell death processes are progressively inactivated during malignant development, in part by loss of tumor suppressors that can promote cell death. The Bin1 gene encodes a nucleocytosolic adaptor protein with tumor suppressor properties, initially identified through its ability to interact with and inhibit malignant transformation by c-Myc and other oncogenes. Bin1 is frequently missing or functionally inactivated in breast and prostate cancers and in melanoma. In this study, we show that Bin1 engages a caspase-independent cell death process similar to type II apoptosis, characterized by cell shrinkage, substratum detachment, vacuolated cytoplasm, and DNA degradation. Cell death induction was relieved by mutation of the BAR domain, a putative effector domain, or by a missplicing event that occurs in melanoma and inactivates suppressor activity. Cells in all phases of the cell cycle were susceptible to death and p53 and Rb were dispensable. Notably, Bin1 did not activate caspases and the broad spectrum caspase inhibitor ZVAD.fmk did not block cell death. Consistent with the lack of caspase involvement, dying cells lacked nucleosomal DNA cleavage and nuclear lamina degradation. Moreover, neither Bcl-2 or dominant inhibition of the Fas pathway had any effect. In previous work, we showed that Bin1 could not suppress cell transformation by SV40 large T antigen. Consistent with this finding, we observed that T antigen suppressed the death program engaged by Bin1. This observation was interesting in light of emerging evidence that T antigen has roles in cell immortalization and human cell transformation beyond Rb and p53 inactivation. In support of a link to c-Myc-induced death processes, AEBSF, a serine protease inhibitor that inhibits apoptosis by c-Myc, potently suppressed DNA degradation by Bin1. Our findings suggest that the tumor suppressor activity of Bin1 reflects engagement of a unique cell death program. We propose that loss of Bin1 may promote malignancy by

  2. Multiple alphaII-spectrin breakdown products distinguish calpain and caspase dominated necrotic and apoptotic cell death pathways.

    PubMed

    Zhang, Zhiqun; Larner, Stephen F; Liu, Ming Cheng; Zheng, Wenrong; Hayes, Ronald L; Wang, Kevin K W

    2009-11-01

    Apoptosis and oncotic necrosis in neuronal and glial cells have been documented in many neurological diseases. Distinguishing between these two major types of cell death in different neurological diseases is needed in order to better reveal the injury mechanisms so as to open up opportunities for therapy development. Accumulating evidence suggests apoptosis and oncosis epitomize the extreme ends of a broad spectrum of morphological and biochemical events. Biochemical markers that can distinguish between the calpain and caspase dominated types of cell death would help in this process. In this study, three chemical agents, maitotoxin (MTX), staurosporine (STS) and thylenediaminetetraacetic acid (EDTA), were used to induce different types of cell death in PC12 neuronal-like cells. MTX-induced necrosis, as determined by the increased levels of calpain-specific cleaved fragments of spectrin by antibodies specific to the calpain-cleaved 150 kDa alphaII-spectrin breakdown product (SBDP150) and 145 kDa alphaII-spectrin breakdown product (SBDP145). In this paradigm, there were no detectable SBDP150i and SBDP120 fragments as determined by antibodies specific to the caspase-cleaved specific fragments similar to those seen in the EDTA-mediated apoptotic PC-12 cells. In contrast to the calpain specific MTX necrosis treatment and the caspase EDTA apoptotic treatment is the STS treatment which induced both proteases as shown by the increase in all the SBDP fragments. Furthermore, compared to SBDP150, SBDP145 appears to be a more specific and sensitive biomarker for calpain activation. Taken together, our results suggested calpains and caspases which dominate the two major types of cell death could be independently discriminated by specifically examining the multiple alphaII-spectrin cleavage breakdown products.

  3. Berberine Induces Caspase-Independent Cell Death in Colon Tumor Cells through Activation of Apoptosis-Inducing Factor

    PubMed Central

    Wang, Lihong; Liu, Liping; Shi, Yan; Cao, Hanwei; Chaturvedi, Rupesh; Calcutt, M. Wade; Hu, Tianhui; Ren, Xiubao; Wilson, Keith T.; Polk, D. Brent; Yan, Fang

    2012-01-01

    Berberine, an isoquinoline alkaloid derived from plants, is a traditional medicine for treating bacterial diarrhea and intestinal parasite infections. Although berberine has recently been shown to suppress growth of several tumor cell lines, information regarding the effect of berberine on colon tumor growth is limited. Here, we investigated the mechanisms underlying the effects of berberine on regulating the fate of colon tumor cells, specifically the mouse immorto-Min colonic epithelial (IMCE) cells carrying the Apcmin mutation, and of normal colon epithelial cells, namely young adult mouse colonic epithelium (YAMC) cells. Berberine decreased colon tumor colony formation in agar, and induced cell death and LDH release in a time- and concentration-dependent manner in IMCE cells. In contrast, YAMC cells were not sensitive to berberine-induced cell death. Berberine did not stimulate caspase activation, and PARP cleavage and berberine-induced cell death were not affected by a caspase inhibitor in IMCE cells. Rather, berberine stimulated a caspase-independent cell death mediator, apoptosis-inducing factor (AIF) release from mitochondria and nuclear translocation in a ROS production-dependent manner. Amelioration of berberine-stimulated ROS production or suppression of AIF expression blocked berberine-induced cell death and LDH release in IMCE cells. Furthermore, two targets of ROS production in cells, cathepsin B release from lysosomes and PARP activation were induced by berberine. Blockage of either of these pathways decreased berberine-induced AIF activation and cell death in IMCE cells. Thus, berberine-stimulated ROS production leads to cathepsin B release and PARP activation-dependent AIF activation, resulting in caspase-independent cell death in colon tumor cells. Notably, normal colon epithelial cells are less susceptible to berberine-induced cell death, which suggests the specific inhibitory effects of berberine on colon tumor cell growth. PMID:22574158

  4. TRAIL-Induced Caspase Activation Is a Prerequisite for Activation of the Endoplasmic Reticulum Stress-Induced Signal Transduction Pathways.

    PubMed

    Lee, Dae-Hee; Sung, Ki Sa; Guo, Zong Sheng; Kwon, William Taehyung; Bartlett, David L; Oh, Sang Cheul; Kwon, Yong Tae; Lee, Yong J

    2016-05-01

    It is well known that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis can be initially triggered by surface death receptors (the extrinsic pathway) and subsequently amplified through mitochondrial dysfunction (the intrinsic pathway). However, little is known about signaling pathways activated by the TRAIL-induced endoplasmic reticulum (ER) stress response. In this study, we report that TRAIL-induced apoptosis is associated with the endoplasmic reticulum (ER) stress response. Human colorectal carcinoma HCT116 cells were treated with TRAIL and the ER stress-induced signal transduction pathway was investigated. During TRAIL treatment, expression of ER stress marker genes, in particular the BiP (binding immunoglobulin protein) gene, was increased and activation of the PERK (PKR-like ER kinase)-eIF2α (eukaryotic initiation factor 2α)-ATF4 (activating transcription factor 4)-CHOP (CCAAT-enhancer-binding protein homologous protein) apoptotic signal transduction pathway occurred. Experimental data from use of a siRNA (small interfering RNA) technique, caspase inhibitor, and caspase-3-deficient cell line revealed that TRAIL-induced caspase activation is a prerequisite for the TRAIL-induced ER stress response. TRAIL-induced ER stress was triggered by caspase-8-mediated cleavage of BAP31 (B cell receptor-associated protein 31). The involvement of the proapoptotic PERK-CHOP pathway in TRAIL-induced apoptosis was verified by using a PERK knockout (PERK(-/-)) mouse embryo fibroblast (MEF) cell line and a CHOP(-/-) MEF cell line. These results suggest that TRAIL-induced the activation of ER stress response plays a role in TRAIL-induced apoptotic death.

  5. Dieldrin promotes proteolytic cleavage of poly(ADP-ribose) polymerase and apoptosis in dopaminergic cells: protective effect of mitochondrial anti-apoptotic protein Bcl-2.

    PubMed

    Kitazawa, Masashi; Anantharam, Vellareddy; Kanthasamy, Arthi; Kanthasamy, Anumantha G

    2004-06-01

    Previously, we demonstrated that the organochlorine pesticide dieldrin induces mitochondrial depolarization, caspase-3 activation and apoptosis in dopaminergic PC12 cells. We also demonstrated that protein kinase Cdelta (PKCdelta), a member of a novel PKC family of proteins, is proteolytically activated by caspase-3 to mediate apoptotic cell death processes. In the present study, we have further characterized the protective effect of the major mitochondrial anti-apoptotic protein Bcl-2 against dieldrin-induced apoptotic events in dopaminergic cells. Exposure to dieldrin (30-100 microM) produced significant cytotoxicity and caspase-3 activation within 3h in vector-transfected PC12 cells, whereas human Bcl-2-transfected PC12 cells were almost completely resistant to dieldrin-induced cytotoxicity and caspase-3 activation. Also, dieldrin (30-300 microM) treatment induced proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), which was blocked by pretreatment with caspase-3 inhibitors Z-DEVD-FMK and Z-VAD-FMK. Additionally, dieldrin-induced chromatin condensation and DNA fragmentation were completely blocked in Bcl-2-overexpressed PC12 cells as compared to vector control cells. Together, these results clearly indicate that overexpression of mitochondrial anti-apoptotic protein protects against dieldrin-induced apoptotic cell death and further suggest that dieldrin primarily alters mitochondrial function to initiate apoptotic cell death in dopaminergic cells.

  6. Radiation-Induced RhoGDIβ Cleavage Leads to Perturbation of Cell Polarity: A Possible Link to Cancer Spreading.

    PubMed

    Fujiwara, Mamoru; Okamoto, Mayumi; Hori, Masato; Suga, Hiroshi; Jikihara, Hiroshi; Sugihara, Yuka; Shimamoto, Fumio; Mori, Toshio; Nakaoji, Koichi; Hamada, Kazuhiko; Ota, Takahide; Wiedemuth, Ralf; Temme, Achim; Tatsuka, Masaaki

    2016-11-01

    The equilibrium between proliferation and apoptosis is tightly balanced to maintain tissue homeostasis in normal tissues and even in tumors. Achieving and maintaining such a balance is important for cancer regrowth and spreading after cytotoxic treatments. Caspase-3 activation and tumor cell death following anticancer therapy as well as accompanying cell death pathways are well characterized, but their association to homeostasis of cancerous tissue and tumor progression remains poorly understood. Here we proposed a novel mechanism of cancer spreading induced by caspase-3. RhoGDIβ, known as a direct cleavage substrate of caspase-3, is overexpressed in many epithelial cancers. The N-terminal-truncated RhoGDIβ (ΔN-RhoGDIβ) is accumulated in caspase-3-activated cells. Stable expression of ΔN-RhoGDIβ in HeLa cells did not induce apoptosis, but impaired directional cell migration in a wound-healing assay accompanied by a perturbed direction of cell division at the wound edge. Subcellular protein fractionation experiments revealed that ΔN-RhoGDIβ but not wild-type RhoGDIβ was present in the detergent-soluble cytoplasmic and nuclear fractions and preferentially associated with Cdc42. Furthermore, Cdc42 activity was constitutively inhibited by stable expression of ΔN-RhoGDIβ, resulting in increased radiation-induced compensatory proliferation linking to RhoA activation. Thus, ΔN-RhoGDIβ dominant-negatively regulates Cdc42 activity and contributes to loss of polarity-related functions. The caspase-3-cleaved RhoGDIβ is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage. J. Cell. Physiol. 231: 2493-2505, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3.

    PubMed

    Espín, Raquel; Roca, Francisco J; Candel, Sergio; Sepulcre, María P; González-Rosa, Juan M; Alcaraz-Pérez, Francisca; Meseguer, José; Cayuela, María L; Mercader, Nadia; Mulero, Victoriano

    2013-03-01

    Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.

  8. Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

    PubMed

    Ruocco, Nadia; Varrella, Stefano; Romano, Giovanna; Ianora, Adrianna; Bentley, Matt G; Somma, Domenico; Leonardi, Antonio; Mellone, Stefano; Zuppa, Antonio; Costantini, Maria

    2016-07-01

    Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Blocking caspase-3-dependent pathway preserves hair cells from salicylate-induced apoptosis in the guinea pig cochlea.

    PubMed

    Feng, Hao; Yin, Shi-Hua; Tang, An-Zhou

    2011-07-01

    In the present study, we aim to explore whether the caspase-3-dependent pathway is involved in the apoptotic cell death that occurs in the hair cells (HCs) of guinea pig cochlea following a salicylate treatment. Guinea pigs received sodium salicylate (Na-SA), at a dose of 200 mg·kg(-1)·d(-1) i.p., as a vehicle for 5 consecutive days. In some experiments, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-FMK), a specific apoptosis inhibitor, was directly applied into the cochlea via the round window niche (RWN) prior to salicylate treatment for determination of caspase-3 activation. Alterations in auditory function were evaluated with auditory brainstem responses (ABR) thresholds. Caspase-3 activity was determined by measuring the proteolytic cleavage product of caspase-3 (N-terminated peptide substrate). DNA fragmentation within the nuclei was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Ultrastructure variation in the target cell was assessed by electron microscopy (EM). Salicylate treatment initiated an obvious elevation in ABR thresholds with a maximum average shift of 60 dB sound pressure level (SPL), and caused significant apoptosis in both inner (IHCs) and outer (OHCs) hair cells resulted from an evident increasing in immunoreactivity to caspase-3 protease. Transmission electron microscopy (TEM) displayed chromatin condensation and nucleus margination accompanied by cell body shrinkage in the OHCs, but not in the IHCs. Scanning electron microscopy (SEM) showed breakdown, fusion, and loss in the stereociliary bundles at the apex of OHCs rather than IHCs. zDEVD-FMK pretreatment prior to salicylate injection substantially attenuated an expression of the apoptotic protease and protected HCs against apoptotic death, followed by a moderate relief in the thresholds of ABR, an alleviation in the submicroscopic structure was also identified. In particular, disorientation and insertion in the

  10. GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway

    PubMed Central

    Rayman, Patricia; Chahlavi, Ali; Ko, Jennifer; Bhattacharjee, Ashish; Li, Yu-Teh; Li, Yuntao; Das, Tanya; Sa, Gaurisankar; Raychaudhuri, Baisakhi; Vogelbaum, Michael A.; Tannenbaum, Charles; Finke, James H.; Biswas, Kaushik

    2015-01-01

    Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases. PMID:26226135

  11. Isoflavone Attenuates the Caspase-1 and Caspase-3 Level in Cell Model of Parkinsonism

    PubMed Central

    Xu, Jian-xin; Song, Hai-ping; Bu, Qing-Xia; Feng, De-Peng; Xu, Xiao-Fan; Sun, Qian-Ru; Li, Xue-Li

    2015-01-01

    The study has investigated the effect of isoflavone attenuates the caspase-1 and caspase-3 level in cell model of Parkinsonism. The subjects were PC12 cells. They were randomly divided into six groups: control, MPP+ (250 μmol/L), isoflavone (10 μM), isoflavone (10 μM) + MPP+ (250 μmol/L), Z-YVAD-CHO (10 nM) + MPP+ group, and Z-DEVD-CHO (10 nM) + MPP+ group. Cell viability was measured by MTT methods; the content of tyrosine hydroxylase was measured by immunocytochemistry method of avidinbiotin peroxidase complex; apoptosis ratio was measured by flow cytometry. The results showed that cell viability in the MPP+ group was lower than in all other five groups. There was no difference in cell viability between isoflavone + MPP+ and control group. Optical density of TH positive cells in isoflavone group was higher than in control, isoflavone + MPP+, and MPP+ only groups. The apoptosis ratio in the isoflavone + MPP+ group and control group and the Z-YVAD-CHO + MPP+ and Z-DEVD-CHO + MPP+ group was similar, which was lower than in the MPP+ group. The lowest apoptosis ratio was found in the isoflavone only group. PMID:26161002

  12. Alpha and omega of carotenoid cleavage.

    PubMed

    Lakshman, M R

    2004-01-01

    In early 1900s, based on indirect evidence, Steenbock and Morton independently predicted that beta-carotene could be the biological precursor of vitamin A, although this notion was contested by others. In the 1930s, Thomas Moore showed the in vivo formation of vitamin A from beta-carotene. But it was not until Jim Olson and DeWitt Goodman independently showed in 1965 the formation of retinal, the aldehyde form of vitamin A from beta-carotene in cell-free extracts of liver and intestine, that this vital pathway of beta-carotene was recognized. Despite compelling evidence in several experimental systems for the central cleavage of beta-carotene to retinal by many investigators, there were some careful independent studies by Glover et al., Ganguly et al., Hansen and Meret and Krinsky et al. showing the eccentric cleavage of beta-carotene resulting in the formation of apocarotenoids both in vivo and in vitro. In an attempt to resolve this controversial issue, we revisited this problem in 1989 and showed beyond doubt the formation of retinal as the sole enzymatic product of a cytosolic enzyme from rabbit and rat intestinal mucosa by mass spectrometry and tracer analysis of the crystallized product. This was confirmed in 1996 by Nagao using the pig intestinal extract. Yeum et al. confirmed in 2000 that retinal is the sole product of beta-carotene cleavage in the presence of alpha-tocopherol, and that the observed formation of apocarotenoids occurs only in the absence of an antioxidant like alpha-tocopherol. In the same year, Barua and Olson also concluded from their in vivo studies in rats that central cleavage is by far the major pathway for the formation of vitamin A from beta-carotene. Beta, beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) is the key enzyme that cleaves beta-carotene into two molecules of retinal. It is a cytosolic enzyme primarily localized in the duodenal mucosa although it has been found in liver. It is a 66 kDa sulfhydryl protein, requires

  13. Non-steroidal Anti-inflammatory Drugs Are Caspase Inhibitors.

    PubMed

    Smith, Christina E; Soti, Subada; Jones, Torey A; Nakagawa, Akihisa; Xue, Ding; Yin, Hang

    2017-02-15

    Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world. While the role of NSAIDs as cyclooxygenase (COX) inhibitors is well established, other targets may contribute to anti-inflammation. Here we report caspases as a new pharmacological target for NSAID family drugs such as ibuprofen, naproxen, and ketorolac at physiologic concentrations both in vitro and in vivo. We characterize caspase activity in both in vitro and in cell culture, and combine computational modeling and biophysical analysis to determine the mechanism of action. We observe that inhibition of caspase catalysis reduces cell death and the generation of pro-inflammatory cytokines. Further, NSAID inhibition of caspases is COX independent, representing a new anti-inflammatory mechanism. This finding expands upon existing NSAID anti-inflammatory behaviors, with implications for patient safety and next-generation drug design.

  14. The executioners sing a new song: killer caspases activate microglia

    PubMed Central

    Venero, J L; Burguillos, M A; Brundin, P; Joseph, B

    2011-01-01

    Activation of microglia and inflammation-mediated neurotoxicity are suggested to have key roles in the pathogenesis of several neurodegenerative disorders. We recently published an article in Nature revealing an unexpected role for executioner caspases in the microglia activation process. We showed that caspases 8 and 3/7, commonly known to have executioner roles for apoptosis, can promote microglia activation in the absence of death. We found these caspases to be activated in microglia of PD and AD subjects. Inhibition of this signaling pathway hindered microglia activation and importantly reduced neurotoxicity in cell and animal models of disease. Here we review evidence suggesting that microglia can have a key role in the pathology of neurodegenerative disorders. We discuss possible underlying mechanisms regulating their activation and neurotoxic effect. We focus on the provocative hypothesis that caspase inhibition can be neuroprotective by targeting the microglia rather than the neurons themselves. PMID:21836616

  15. ROFA INCREASES CASPASE-3 ACTIVITY IN HUMAN ALVEOLAR MACRAPHAGE

    EPA Science Inventory

    Exposure to air pollution particles produces pulmonary inflammation and injury, but the mechanisms of this injury are unclear. Apoptosis, involving activation of caspases, may be one potential mechanism. In this study, we hypothesized that ROFA, a constituent of air pollution...

  16. Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells.

    PubMed

    Chornyy, Sergiy; Parkhomenko, Julia; Chorna, Nataliya

    2007-01-01

    Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.

  17. Environmental neurotoxin dieldrin induces apoptosis via caspase-3-dependent proteolytic activation of protein kinase C delta (PKCdelta): Implications for neurodegeneration in Parkinson's disease

    PubMed Central

    Kanthasamy, Anumantha G; Kitazawa, Masashi; Yang, Yongjie; Anantharam, Vellareddy; Kanthasamy, Arthi

    2008-01-01

    Background In previous work, we investigated dieldrin cytotoxicity and signaling cell death mechanisms in dopaminergic PC12 cells. Dieldrin has been reported to be one of the environmental factors correlated with Parkinson's disease and may selectively destroy dopaminergic neurons. Methods Here we further investigated dieldrin toxicity in a dopaminergic neuronal cell model of Parkinson's disease, namely N27 cells, using biochemical, immunochemical, and flow cytometric analyses. Results In this study, dieldrin-treated N27 cells underwent a rapid and significant increase in reactive oxygen species followed by cytochrome c release into cytosol. The cytosolic cytochrome c activated caspase-dependent apoptotic pathway and the increased caspase-3 activity was observed following a 3 hr dieldrin exposure in a dose-dependent manner. Furthermore, dieldrin caused the caspase-dependent proteolytic cleavage of protein kinase C delta (PKCδ) into 41 kDa catalytic and 38 kDa regulatory subunits in N27 cells as well as in brain slices. PKCδ plays a critical role in executing the apoptotic process in dieldrin-treated dopaminergic neuronal cells because pretreatment with the PKCδ inhibitor rottlerin, or transfection and over-expression of catalytically inactive PKCδK376R, significantly attenuates dieldrin-induced DNA fragmentation and chromatin condensation. Conclusion Together, we conclude that caspase-3-dependent proteolytic activation of PKCδ is a critical event in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells. PMID:18945348

  18. Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis.

    PubMed

    Nath, R; Huggins, M; Glantz, S B; Morrow, J S; McGinnis, K; Nadimpalli, R; Wanga, K K

    2000-10-01

    Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478)*SVEAL). Here, by using a synthetic peptide (NH(2)-SVEALC) mimicking the neo-N-terminal of SBDP120 as antigen, we report the development of chicken antibodies that specifically recognize the SBDP120 generated by in vitro caspase-3 digestion of bovine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to human neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-spectrin nor its other fragments on Western blots. These anti-SBDP120 work equally well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical studies, these antibodies also specifically stained apoptotic SH-SY5Y or CGN's undergoing apoptosis in a caspase- inhibitor-sensitive manner. These anti-SBDP120s might become powerful markers for apoptotic neurons in various neurological or neurodegenerative conditions in vivo.

  19. Squamous Cell Carcinoma Antigen 1 Promotes Caspase-8-Mediated Apoptosis in Response to Endoplasmic Reticulum Stress While Inhibiting Necrosis Induced by Lysosomal Injury▿

    PubMed Central

    Ullman, Erica; Pan, Ji-An; Zong, Wei-Xing

    2011-01-01

    Squamous cell carcinoma antigen 1 (SCCA1) is a member of the serine protease inhibitor (serpin) family of proteins, whose target proteases include the cathepsins. Initially identified as a serological marker for advanced squamous cell carcinomas of the cervix, SCCA1 has also been found to be associated with other cancer types of epithelial or endodermal origins such as lung cancer, head and neck cancer, melanoma, and hepatocellular carcinoma. While the biological function of SCCA1 remains largely unclear, it is believed to limit cellular damage resulting from lysosomal cathepsin release. Here, we show that SCCA1 acts as a molecular switch that inhibits cell death induced by lysosomal injury resulting from DNA alkylating agents and hypotonic shock, whereas it promotes a caspase-8-mediated apoptosis in response to endoplasmic reticulum (ER) stress. In response to ER stress, SCCA1 blocks both lysosomal and proteasomal protein degradation pathways and enhances the interaction between sequestosome 1/p62 and caspase-8, which leads to the aggregation of intracellular caspase-8 and its subsequent cleavage and activation. Hence, on one hand, SCCA1 inhibits cell death induced by lysosomal injury while, on the other hand, it sensitizes cells to ER stress by activating caspase-8 independently of the death receptor apoptotic pathway. PMID:21576355

  20. Pinus densiflora leaf essential oil induces apoptosis via ROS generation and activation of caspases in YD-8 human oral cancer cells

    PubMed Central

    JO, JEONG-RANG; PARK, JU SUNG; PARK, YU-KYOUNG; CHAE, YOUNG ZOO; LEE, GYU-HEE; PARK, GY-YOUNG; JANG, BYEONG-CHURL

    2012-01-01

    The leaf of Pinus (P.) densiflora, a pine tree widely distributed in Asian countries, has been used as a traditional medicine. In the present study, we investigated the anticancer activity of essential oil, extracted by steam distillation, from the leaf of P. densiflora in YD-8 human oral squamous cell carcinoma (OSCC) cells. Treatment of YD-8 cells with P. densiflora leaf essential oil (PLEO) at 60 μg/ml for 8 h strongly inhibited proliferation and survival and induced apoptosis. Notably, treatment with PLEO led to generation of ROS, activation of caspase-9, PARP cleavage, down-regulation of Bcl-2, and phosphorylation of ERK-1/2 and JNK-1/2 in YD-8 cells. Treatment with PLEO, however, did not affect the expression of Bax, XIAP and GRP78. Importantly, pharmacological inhibition studies demonstrated that treatment with vitamin E (an anti-oxidant) or z-VAD-fmk (a pan-caspase inhibitor), but not with PD98059 (an ERK-1/2 inhibitor) or SP600125 (a JNK-1/2 inhibitor), strongly suppressed PLEO-induced apoptosis in YD-8 cells and reduction of their survival. Vitamin E treatment further blocked activation of caspase-9 and Bcl-2 down-regulation induced by PLEO. Thus, these results demonstrate firstly that PLEO has anti-proliferative, anti-survival and pro-apoptotic effects on YD-8 cells and the effects are largely due to the ROS-dependent activation of caspases. PMID:22086183

  1. Cutting edge: IL-1β processing during Pseudomonas aeruginosa infection is mediated by neutrophil serine proteases and is independent of NLRC4 and caspase-1.

    PubMed

    Karmakar, Mausita; Sun, Yan; Hise, Amy G; Rietsch, Arne; Pearlman, Eric

    2012-11-01

    To examine the role of caspase-1 and the NLRC4 inflammasome during bacterial infection, C57BL/6, IL-1β(-/-), caspase-1(-/-), and NLRC4(-/-) mouse corneas were infected with ExoS/T- or ExoU-expressing Pseudomonas aeruginosa. We found that IL-1β was essential for neutrophil recruitment and bacterial clearance and was produced by myeloid cells rather than resident cells. In addition, neutrophils were found to be the primary source of mature IL-1β during infection, and there was no significant difference in IL-1β processing between C57BL/6 and caspase-1(-/-) or NLRC4(-/-) infected corneas. IL-1β cleavage by human and mouse neutrophils was blocked by serine protease inhibitors and was impaired in infected neutrophil elastase (NE)(-/-) corneas. NE(-/-) mice also had an impaired ability to clear the infection. Together, these results demonstrate that during P. aeruginosa infection, neutrophils are the primary source of mature IL-1β and that IL-1β processing is dependent on serine proteases and not NLRC4 or caspase-1.

  2. Environmental neurotoxin dieldrin induces apoptosis via caspase-3-dependent proteolytic activation of protein kinase C delta (PKCdelta): Implications for neurodegeneration in Parkinson's disease.

    PubMed

    Kanthasamy, Anumantha G; Kitazawa, Masashi; Yang, Yongjie; Anantharam, Vellareddy; Kanthasamy, Arthi

    2008-10-22

    In previous work, we investigated dieldrin cytotoxicity and signaling cell death mechanisms in dopaminergic PC12 cells. Dieldrin has been reported to be one of the environmental factors correlated with Parkinson's disease and may selectively destroy dopaminergic neurons. Here we further investigated dieldrin toxicity in a dopaminergic neuronal cell model of Parkinson's disease, namely N27 cells, using biochemical, immunochemical, and flow cytometric analyses. In this study, dieldrin-treated N27 cells underwent a rapid and significant increase in reactive oxygen species followed by cytochrome c release into cytosol. The cytosolic cytochrome c activated caspase-dependent apoptotic pathway and the increased caspase-3 activity was observed following a 3 hr dieldrin exposure in a dose-dependent manner. Furthermore, dieldrin caused the caspase-dependent proteolytic cleavage of protein kinase C delta (PKCδ) into 41 kDa catalytic and 38 kDa regulatory subunits in N27 cells as well as in brain slices. PKCδ plays a critical role in executing the apoptotic process in dieldrin-treated dopaminergic neuronal cells because pretreatment with the PKCδ inhibitor rottlerin, or transfection and over-expression of catalytically inactive PKCδ(K)³⁷⁶(R), significantly attenuates dieldrin-induced DNA fragmentation and chromatin condensation. Together, we conclude that caspase-3-dependent proteolytic activation of PKCδ is a critical event in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells.

  3. Methyl methanesulfonate induces apoptosis in p53-deficient H1299 and Hep3B cells through a caspase 2- and mitochondria-associated pathway.

    PubMed

    Jiang, Ying; Zhang, Xiao-Yun; Sun, Li; Zhang, Guang-Lin; Duerksen-Hughes, Penelope; Zhu, Xin-Qiang; Yang, Jun

    2012-11-01

    Methyl methanesulfonate (MMS) has been shown to induce apoptosis in various cell types through p53-dependent pathways. Nevertheless, pharmacological and genetic blockade of p53 functions results in similar or delayed sensitivity to MMS treatment, suggesting the presence of p53-independent apoptotic mechanisms. To understand the p53-independent mechanisms that are engaged during MMS-induced apoptosis, we established MMS-induced apoptotic cell models using p53-deficient H1299 and Hep3B cells. Our results demonstrated that MMS at concentrations of 50, 100, 200, 400 and 800 μM induced the formation of gammaH2AX foci, and that at higher concentrations, 400 and 800 μM, MMS treatment led to apoptosis in the two cell lines. This apoptotic cell death was concurrent with the loss of mitochondrial membrane potential, nuclear-cytosolic translocation of active caspase 2, release of cytochrome c from mitochondria, and the cleavage of caspase 9, caspase 3 and PARP. However, MMS-induced DNA damage failed to stabilize the p53 family members TAp73 and DNp73. These results demonstrated a p53- and p73-independent mechanism for MMS-induced apoptosis that involves the nuclear-cytosolic translocation of active caspase 2 as well as the mitochondria-mediated pathway.

  4. Cleavage crystallography of liquid metal embrittled aluminum alloys

    NASA Technical Reports Server (NTRS)

    Reynolds, A. P.; Stoner, G. E.

    1991-01-01

    The crystallography of liquid metal-induced transgranular cleavage in six aluminum alloys having a variety of microstructures has been determined via Laue X-ray back reflection. The cleavage crystallography was independent of alloy microstructure, and the cleavage plane was 100-plane oriented in all cases. It was further determined that the cleavage crystallography was not influenced by alloy texture. Examination of the fracture surface indicated that there was not a unique direction of crack propagation. In addition, the existence of 100-plane cleavage on alloy 2024 fracture surfaces was inferred by comparison of secondary cleavage crack intersection geometry on the 2024 surfaces with the geometry of secondary cleavage crack intersections on the test alloys.

  5. Caspase Deficiency: Involvement in Breast Carcinogenesis and Resistance

    DTIC Science & Technology

    2001-07-01

    caspase 3 cleave a number of cellular death substrates, such as poly ADP-ribose polymerase (PARP), DNA fragmentation factor (DFF), protein kinase c and...1996. 16 16. Janicke, R. U., Sprengart, M. L., Wati, M. R., and Porter, A. G. Caspase-3 is required for DNA fragmentation and morphological changes...because they cleave cellular substrates, including poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, DNA fragmentation factor, lamin 11

  6. Caspase Deficiency: Involvement in Breast Carcinogenesis and Resistance

    DTIC Science & Technology

    2003-07-01

    triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors . Cancer Res. 1997 Nov 1;57(21):4956-64. 4...process involved in homeostasis and the biochemical responses to different anti- tumor therapies. Aberrant expression of other apoptotic regulators...response to several anti-cancer agents, including chemotherapy, radiation- and tumor necrosis factor alpha (TNF-a). Caspase 3 was expressed in MCF-7 cells by

  7. Increased caspase-3 immunoreactivity of erythrocytes in STZ diabetic rats.

    PubMed

    Fırat, Uğur; Kaya, Savaş; Cim, Abdullah; Büyükbayram, Hüseyin; Gökalp, Osman; Dal, Mehmet Sinan; Tamer, Mehmet Numan

    2012-01-01

    Eryptosis is a term to define apoptosis of erythrocytes. Oxidative stress and hyperglycemia, both of which exist in the diabetic intravascular environment, can trigger eryptosis of erythrocytes. In this experimental study, it is presented that the majority of erythrocytes shows caspase-3 immunoreactivity in streptozocin- (STZ)-induced diabetic rats. Besides that, caspase-3 positive erythrocytes are aggregated and attached to vascular endothelium. In conclusion, these results may start a debate that eryptosis could have a role in the diabetic complications.

  8. Cleavage of Interferon Regulatory Factor 7 by Enterovirus 71 3C Suppresses Cellular Responses

    PubMed Central

    Lei, Xiaobo; Xiao, Xia; Xue, Qinghua; Jin, Qi

    2013-01-01

    Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses. PMID:23175366

  9. Evidence of the involvement of caspase-1 under physiologic and pathologic cellular stress during human pregnancy: a link between the inflammasome and parturition.

    PubMed

    Gotsch, Francesca; Romero, Roberto; Chaiworapongsa, Tinnakorn; Erez, Offer; Vaisbuch, Edi; Espinoza, Jimmy; Kusanovic, Juan Pedro; Mittal, Pooja; Mazaki-Tovi, Shali; Kim, Chong Jai; Kim, Jung Sun; Edwin, Samuels; Nhan-Chang, Chia-Ling; Hamill, Neil; Friel, Laraa; Than, Nandor Gabor; Mazor, Moshe; Yoon, Bo Hyun; Hassan, Sonia S

    2008-09-01

    Caspase-1 is a component of the NALP3 inflammasome, a cytosolic multiprotein complex that mediates the processing of pro-inflammatory caspases and cytokines. The inflammasome represents the first line of defense against cellular stress and is a crucial component of innate immunity. Caspase-1 is the enzyme responsible for the cleavage and activation of interleukin (IL)-1 beta, which is a potent pro-inflammatory cytokine, and plays a central role in the mechanisms leading to labor (preterm and term) particularly in the context of intrauterine infection/inflammation. In addition, caspase-1 cleaves IL-18 and IL-33. The objectives of this study were to determine whether there is a relationship between amniotic fluid concentrations of caspase-1 and gestational age, parturition (term and preterm), and intra-amniotic infection/inflammation (IAI). A cross-sectional study was conducted including 143 pregnant women in the following groups: (1) mid-trimester of pregnancy (n = 18); (2) term not in labor (n = 25); (3) term in labor (n = 28); (4) preterm labor (PTL) who delivered at term (n = 23); (5) PTL without IAI who delivered preterm (n = 32); (6) PTL with IAI who delivered preterm neonates (n = 17). Caspase-1 concentrations in amniotic fluid were determined by a specific and sensitive immunoassay. Non-parametric statistics were used for analysis. (1) Caspase-1 was detected in amniotic fluid of women at term, but in none of the mid-trimester samples. (2) Patients in labor at term had a significantly higher median amniotic fluid concentration of caspase-1 than women at term not in labor (term in labor: 10.5 pg/mL, range 0.0-666.0 vs. term not in labor: 5.99 pg/mL, range 0.0-237.4; p < 0.05). (3) Among patients with spontaneous PTL, those with IAI (median 41.4 pg/mL, range 0.0-515.0) had a significantly higher median amniotic fluid caspase-1 concentration than those without IAI who delivered preterm (median 0.0 pg/mL, range 0.0-78.4) and than those who delivered at term

  10. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine

    PubMed Central

    Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C.

    2015-01-01

    Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases. PMID:26400108

  11. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine.

    PubMed

    Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C

    2015-09-24

    Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

  12. Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line.

    PubMed

    Xiao, Hui; Rawal, Malika; Hahm, Eun-Ryeong; Singh, Shivendra V

    2007-11-15

    We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, downregulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.

  13. NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - But not palmitate-induced toxicity.

    PubMed

    Anderson, Kimberley J; Russell, Aaron P; Foletta, Victoria C

    2015-01-01

    The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

  14. Human caspase-3 inhibition by Z-tLeu-Asp-H: tLeu(P{sub 2}) counterbalances Asp(P{sub 4}) and Glu(P{sub 3}) specific inhibitor truncation

    SciTech Connect

    Colantonio, Patrizia; Leboffe, Loris; Bolli, Alessandro; Marino, Maria; Ascenzi, Paolo; Luisi, Grazia

    2008-12-19

    Caspase-3 is responsible for the cleavage of several proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Designed on the cleavage site of PARP, Ac-Asp-Glu-Val-Asp-H has been reported as a highly specific inhibitor. To overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of tetra-peptidyl aldehydes, di- and tri-peptidyl caspase-3 inhibitors have been synthesized. Here, the synthesis and the inhibition properties of peptidyl aldehydes Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H are reported. Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H inhibit competitively human caspase-3 activity in vitro with K{sub i}{sup 0} = 3.6 nM, 18.2 nM, and 109 nM, respectively (pH 7.4 and 25 deg. C). Moreover, Z-tLeu-Asp-H impairs apoptosis in human DLD-1 colon adenocarcinoma cells without affecting caspase-8. Therefore, Ac-Asp-Glu-Val-Asp-H can be truncated to Z-tLeu-Asp-H retaining nanomolar inhibitory activity in vitro and displaying action in whole cells, these properties reflect the unprecedented introduction of the bulky and lipophilic tLeu residue at the P{sub 2} position.

  15. Tumor necrosis factor-alpha induces apoptosis associated with poly(ADP-ribose) polymerase cleavage in HT-29 colon cancer cells.

    PubMed

    Vaculová, Alena; Hofmanova, Jirina; Soucek, Karel; Kovariková, Martina; Kozubík, Alois

    2002-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is known for its selective cytotoxic activity on tumour cells. We analysed the response of HT-29 human colon carcinoma cells to this cytokine. After TNF-alpha treatment, cell proliferation, cell cycle, reactive oxygen species (ROS) production (flow cytometry), the amount of apoptotic cells (flow cytometry, fluorescence microscopy), cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 activity (Western blotting) were detected. TNF-alpha induced a decrease of cell growth and viability, an accumulation of cells in the S-phase of the cell cycle, an increase of subdiploid cell population and nuclear chromatin condensation and fragmentation, but not sooner than 96-120 hours. However, earlier events characteristic of apoptosis occurred, such as caspase-3 activation, PARP cleavage to 89 kDa fragment and changes in ROS production. We demonstrated that, in addition to being an early marker of apoptosis, activation of caspase-3 and degradation of PARP may play a causative role in HT-29 cell death induced by TNF-alpha.

  16. Caspase-12 ablation preserves muscle function in the mdx mouse

    PubMed Central

    Moorwood, Catherine; Barton, Elisabeth R.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin. Several downstream consequences of dystrophin deficiency are triggers of endoplasmic reticulum (ER) stress, including loss of calcium homeostasis, hypoxia and oxidative stress. During ER stress, misfolded proteins accumulate in the ER lumen and the unfolded protein response (UPR) is triggered, leading to adaptation or apoptosis. We hypothesized that ER stress is heightened in dystrophic muscles and contributes to the pathology of DMD. We observed increases in the ER stress markers BiP and cleaved caspase-4 in DMD patient biopsies, compared with controls, and an increase in multiple UPR pathways in muscles of the dystrophin-deficient mdx mouse. We then crossed mdx mice with mice null for caspase-12, the murine equivalent of human caspase-4, which are resistant to ER stress. We found that deleting caspase-12 preserved mdx muscle function, resulting in a 75% recovery of both specific force generation and resistance to eccentric contractions. The compensatory hypertrophy normally found in mdx muscles was normalized in the absence of caspase-12; this was found to be due to decreased fibre sizes, and not to a fibre type shift or a decrease in fibrosis. Fibre central nucleation was not significantly altered in the absence of caspase-12, but muscle fibre degeneration found in the mdx mouse was reduced almost to wild-type levels. In conclusion, we have identified heightened ER stress and abnormal UPR signalling as novel contributors to the dystrophic phenotype. Caspase-4 is therefore a potential therapeutic target for DMD. PMID:24879640

  17. Novel acylated steroidal glycosides from Caralluma tuberculata induce caspase-dependent apoptosis in cancer cells.

    PubMed

    Waheed, Abdul; Barker, James; Barton, Stephen J; Khan, Gul-Majid; Najm-Us-Saqib, Qazi; Hussain, Manzoor; Ahmed, Sabbir; Owen, Caroline; Carew, Mark A

    2011-10-11

    apoptosis at <25 μM after 48 h as assessed by cell shrinkage, PARP cleavage, DNA fragmentation, and reversal with the caspase inhibitor. Two novel steroid glycosides isolated from Caralluma tuberculata possess moderate, micromolar cytotoxic activity on breast cancer and other cells in vitro, which may indicate a source of activity in vivo of interest to future drug design. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Resistance of Actin to Cleavage during Apoptosis

    NASA Astrophysics Data System (ADS)

    Song, Qizhong; Wei, Tie; Lees-Miller, Susan; Alnemri, Emad; Watters, Dianne; Lavin, Martin F.

    1997-01-01

    A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β -converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

  19. Activity dependent CAM cleavage and neurotransmission

    PubMed Central

    Conant, Katherine; Allen, Megan; Lim, Seung T.

    2015-01-01

    Spatially localized proteolysis represents an elegant means by which neuronal activity dependent changes in synaptic structure, and thus experience dependent learning and memory, can be achieved. In vitro and in vivo studies suggest that matrix metalloproteinase and adamalysin activity is concentrated at the cell surface, and emerging evidence suggests that increased peri-synaptic expression, release and/or activation of these proteinases occurs with enhanced excitatory neurotransmission. Synaptically expressed cell adhesion molecules (CAMs) could therefore represent important targets for neuronal activity-dependent proteolysis. Several CAM subtypes are expressed at the synapse, and their cleavage can influence the efficacy of synaptic transmission through a variety of non-mutually exclusive mechanisms. In the following review, we discuss mechanisms that regulate neuronal activity-dependent synaptic CAM shedding, including those that may be calcium dependent. We also highlight CAM targets of activity-dependent proteolysis including neuroligin and intercellular adhesion molecule-5 (ICAM-5). We include discussion focused on potential consequences of synaptic CAM shedding, with an emphasis on interactions between soluble CAM cleavage products and specific pre- and post-synaptic receptors. PMID:26321910

  20. Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants[OPEN

    PubMed Central

    Li, Yurong; Kabbage, Mehdi; Liu, Wende; Dickman, Martin B.

    2016-01-01

    The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved group of cochaperones that modulate numerous cellular processes. Previously we found that Arabidopsis thaliana BAG6 is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanisms by which BAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for BAG6-mediated basal resistance. We show that Arabidopsis BAG6 is cleaved in vivo in a caspase-1-like-dependent manner and via a combination of pull-downs, mass spectrometry, yeast two-hybrid assays, and chemical genomics, we demonstrate that BAG6 interacts with a C2 GRAM domain protein (BAGP1) and an aspartyl protease (APCB1), both of which are required for BAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that BAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of BAGP1 or APCB1 results in the blocking of BAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage and inhibits autophagy in plants; disease resistance is also compromised. Taken together, these results identify a mechanism that couples an aspartyl protease with a molecular cochaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance. PMID:26739014

  1. Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses

    PubMed Central

    Bulau, Ana-Maria; Nold, Marcel F.; Li, Suzhao; Nold-Petry, Claudia A.; Fink, Michaela; Mansell, Ashley; Schwerd, Tobias; Hong, Jaewoo; Rubartelli, Anna; Dinarello, Charles A.; Bufler, Philip

    2014-01-01

    IL-37 is a fundamental inhibitor of innate immunity. Human IL-37 has a caspase-1 cleavage site and translocates to the nucleus upon LPS stimulation. Here, we investigated whether caspase-1 processing affects IL-37–mediated suppression of LPS-induced cytokines and the release from cells by analyzing a caspase-1 cleavage site mutant IL-37 (IL-37D20A). Nuclear translocation of IL-37D20A is significantly impaired compared with WT IL-37 in transfected cells. LPS-induced IL-6 was decreased in cells expressing WT IL-37 but not IL-37D20A. The function of IL-37 in transfected bone marrow-derived macrophages is nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-dependent, because IL-37 transfection in apoptosis-associated speck-like protein containing a carboxyl-terminal caspase recruitment domain- and NLRP3-deficient cells does not reduce levels of IL-6 and IL-1β upon LPS stimulation. IL-37–expressing macrophages release both precursor and mature IL-37, but only the externalization of mature IL-37 was dependent on ATP. Precursor and mature IL-37 was also secreted from human dendritic cells and peripheral blood mononuclear cells. To determine whether IL-37 is active in the extracellular compartment, we pretreated IL-37 transgenic mice with IL-37–neutralizing antibodies before LPS challenge. In IL-37–expressing mice, neutralizing IL-37 antibodies reversed the suppression of LPS-induced serum IL-6. In contrast, the addition of neutralizing antibody did not reverse suppression of LPS-induced IL-6 in mouse macrophages transfected with IL-37. Although caspase-1 is required for nuclear translocation of intracellular IL-37 and for secretion of mature IL-37, the release of the IL-37 precursor is independent of caspase-1 activation. IL-37 now emerges as a dual-function cytokine with intra- and extracellular properties for suppressing innate inflammation. PMID:24481253

  2. Interferon γ inhibits growth of human pancreatic carcinoma cells via caspase-1 dependent induction of apoptosis

    PubMed Central

    Detjen, K; Farwig, K; Welzel, M; Wiedenmann, B; Rosewicz, S

    2001-01-01

    BACKGROUND AND AIMS—The poor prognosis of pancreatic cancer is partly due to resistance to a broad spectrum of apoptotic stimuli. To identify intact proapoptotic pathways of potential clinical relevance, we characterised the effects of interferon γ (IFN-γ) on growth and survival in human pancreatic cancer cells.
METHODS—IFN-γ receptor expression and signal transduction were examined by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoprecipitation, western blot analysis, and transactivation assays. Effects on cell growth and survival were evaluated in terms of cell numbers, colony formation, cell cycle analysis, DNA fragmentation, and poly(ADP ribose) polymerase (PARP) cleavage.
RESULTS—All four pancreatic cancer cell lines examined expressed functional IFN-γ receptors and downstream effectors, including the putative tumour suppressor interferon regulatory factor 1 (IRF-1). IFN-γ treatment profoundly inhibited anchorage dependent and independent growth of pancreatic cancer cells. Cell cycle analyses revealed subdiploid cells suggesting apoptosis, which was confirmed by demonstration of DNA fragmentation and PARP cleavage. Time and dose dependency of apoptosis induction and growth inhibition correlated closely, identifying apoptosis as the main, if not exclusive, mechanism responsible for growth inhibition. Apoptosis was preceded by upregulation of procaspase-1 and accompanied by proteolytic activation. Furthermore, the caspase inhibitor z-vad-fmk completely prevented IFN-γ mediated apoptosis.
CONCLUSIONS—These results identify an intact proapoptotic pathway in pancreatic cancer cells and suggest that IRF-1 and/or procaspase-1 may represent potential therapeutic targets to be further explored.


Keywords: interferon γ; apoptosis; caspase-1;interferon regulatory factor; pancreatic cancer PMID:11454803

  3. Ceramide mediates caspase-independent programmed cell death.

    PubMed

    Thon, Lutz; Möhlig, Heike; Mathieu, Sabine; Lange, Arne; Bulanova, Elena; Winoto-Morbach, Supandi; Schütze, Stefan; Bulfone-Paus, Silvia; Adam, Dieter

    2005-12-01

    Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase-dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase-independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase-independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor-interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase-independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl-2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.

  4. Energy requirement for caspase activation and neuronal cell death.

    PubMed

    Nicotera, P; Leist, M; Fava, E; Berliocchi, L; Volbracht, C

    2000-04-01

    Recent work has shown that execution of the apoptotic program involves a relatively limited number of pathways. According to a general view, these would converge to activate the caspase family of proteases. However, there is increasing evidence that apoptotic-like features can be found also when cells are treated with inhibitors of caspases as the cell permeable tripeptide, Z-Val-Ala-Asp-fluoro-methyl-ketone (Z-VAD-fmk), or analogous compounds. This has posed the question as to whether apoptosis may occur in a caspase independent way, and whether caspase inhibitors may then be used to treat diseases characterised by an excess apoptosis. It is also becoming clear, that ATP depletion during the early phases of apoptosis can preclude caspase activation, and consequently switch execution of cell death towards necrosis. In vivo, a block or partial inhibition of the typical apoptotic demise may have profound implications, as persistence of damaged but "undead" cells within the nervous system, followed by delayed lysis may favour neuroinflammatory reactions. In this review, we discuss some recent findings, which suggest that cells may use diverging execution pathways, with different implications in neuropathology and therapy.

  5. Early cleavage in Phoronis muelleri (Phoronida) displays spiral features.

    PubMed

    Pennerstorfer, Markus; Scholtz, Gerhard

    2012-01-01

    The view that early cleavage in Phoronida follows a radial pattern is widely accepted. However, data supporting this characterization are ambiguous. Studies have been repeatedly reporting variation between individual embryos, and the occurrence of embryos exhibiting oblique divisions or nonradial cell arrangements. Such embryos were often considered to represent variation within radial cleavage, or artificial appearances. Cleavage in Phoronis muelleri was previously characterized as "derived radial," but also oblique spindles and cell elongations, and shifted cell arrangements were observed. We studied the early cleavage in P. muelleri applying 4D microscopy, fluorescent staining, and confocal laser scanning microscopy. To deal with the problem of variation we provide statistical evaluations of our data. These show that oblique divisions do not represent variational abnormalities. In fact, they reveal that most cells divide obliquely from the third cleavage onwards. What is more, in almost all cells the axis of the third cleavage is inclined dextrally. The fourth cleavage is even stronger sinistrally pronounced. Subsequently, the pattern of alternating cleavage orientation is largely restricted to animal and vegetal blastomeres. As a result of the obliqueness of divisions, four cells encircle the poles in most embryos. Cross furrows are occasionally present. We found no indications for radial cleavage in P. muelleri. In contrast, the observed cleavage displays several characters consistent with the pattern of spiral cleavage. A close relation of phoronid and spiralian cleavage is also suggested by molecular phylogenies, allying both groups in the Lophotrochozoa. We suggest our findings to represent morphological support for this lophotrochozoan/spiralian affinity of Phoronida.

  6. [On the Features of Embryonic Cleavage in Diverse Fish Species].

    PubMed

    Desnitskiy, A G

    2015-01-01

    Literature on the earliest steps of fish embryogenesis (including a number of "non-model" species) has been considered. The main attention has been paid to the loss of cleavage division synchrony and the first latitudinal cleavage furrow. In teleostean embryos, the features of their meroblastic cleavage are not rigidly associated with egg size. The midblastula transition (in a form clearly enough) occurs in some chondrostean and teleostean fishes, but it has not been detected in the representatives of sarcopterygian and chondrichthyan fishes.

  7. Caspase-8 and Caspase-9 Functioned Differently at Different Stages of the Cyclic Stretch-Induced Apoptosis in Human Periodontal Ligament Cells

    PubMed Central

    Zhuang, Jiabao; Zhang, Fuqiang; Xu, Chun

    2016-01-01

    Background Human periodontal ligament (PDL) cells underwent apoptosis after mechanical stretch loading. However, the exact signalling pathway remains unknown. This study aimed to elucidate how the apoptotic caspases functioned in the cyclic stretch-induced apoptosis in human PDL cells. Materials and Methods In the present study, 20% cyclic stretch was selected to load the cells for 6 or 24 h. The following parameters were analyzed: apoptotic rates, the protein levels of caspase-3, -7, -8 and -9 and the activities of caspase-8 and -9. Subsequently, the influences of caspase-8 and caspase-9 inhibitors on the apoptotic rate and the protein level of the activated caspase-3 were assessed as well. Results The apoptotic rates increased in response to cyclic stretch, but the cells entered different apoptotic stages after 6 and 24 h stretches. Caspase-3, -7, -8 and -9 were all activated after stretch loading. The stretch-induced apoptosis and the protein level of the activated caspase-3 were inhibited after inhibiting both caspase-8 and caspase-9 in both 6 and 24 h stretched cells and after inhibiting caspase-9 in 24 h stretched cells. Conclusion Caspase-8 and -9 functioned differently at different apoptotic stages in human PDL cells after cyclic stretch. PMID:27942018

  8. Cytoplasmic myosin exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability

    PubMed Central

    Cui, Xiaoxuan; Zhang, Lu; Magli, Amanda R.; Catera, Rosa; Yan, Xiao-Jie; Griffin, Daniel O.; Rothstein, Thomas L.; Barrientos, Jacqueline; Kolitz, Jonathan E.; Allen, Steven L.; Rai, Kanti R.; Chiorazzi, Nicholas; Chu, Charles C.

    2015-01-01

    The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease. PMID:26220042

  9. A transgenic mouse for imaging caspase-dependent apoptosis within the skin.

    PubMed

    Khanna, Divya; Hamilton, Christin A; Bhojani, Mahaveer S; Lee, Kuei C; Dlugosz, Andrej; Ross, Brian D; Rehemtulla, Alnawaz

    2010-07-01

    Apoptosis is an essential process for the maintenance of normal physiology. The ability to noninvasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of beta-galactosidase gene flanked by two estrogen receptor regulatory domains and intervening Asp-Glu-Val-Glu sequences was constructed to serve as a tool for in vivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable beta-galactosidase activity. Caspase 3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of beta-galactosidase activity. Enzymatic activation of the reporter during an apoptotic event enabled noninvasive measurement of beta-galactosidase activity in living cells, which correlated with traditional measures of apoptosis in a dose- and time-dependent manner. Using a near-infrared fluorescent substrate of beta-galactosidase (9H-{1,3-dichloro-9,9-dimethylacridin-2-one-7-yl} beta-D-galactopyranoside), noninvasive in vivo imaging of apoptosis was achieved in a xenograft tumor model in response to proapoptotic therapy. Finally, a transgenic mouse model was developed expressing the ER-LACZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals, especially in the context of skin biology.

  10. β-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway.

    PubMed

    Yu, Hai Yang; Kim, Sung Ok; Jin, Cheng-Yun; Kim, Gi-Young; Kim, Wun-Jae; Yoo, Young Hyun; Choi, Yung Hyun

    2014-05-01

    β-lapachone is a naturally occurring quinone that selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo; however, its mechanism of action needs to be further elaborated. In this study, we investigated the effects of β-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. β-lapachone significantly inhibited cellular proliferation, and some typical apoptotic characteristics such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells were observed in β-lapachone-treated AGS cells. Treatment with β-lapachone caused mitochondrial transmembrane potential dissipation, stimulated the mitochondria-mediated intrinsic apoptotic pathway, as indicated by caspase-9 activation, cytochrome c release, Bcl-2 downregulation and Bax upregulation, as well as death receptor-mediated extrinsic apoptotic pathway, as indicated by activation of caspase-8 and truncation of Bid. This process was accompanied by activation of caspase-3 and concomitant with cleavage of poly(ADP-ribose) polymerase. The general caspase inhibitor, z-VAD-fmk, significantly abolished β-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by β-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased β-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of β-lapachone is probably regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways, and that inhibition of the PI3K/Akt signaling may contribute to β-lapachone-mediated AGS cell growth inhibition and apoptosis induction.

  11. Destabilization of CARP mRNAs by aloe-emodin contributes to caspase-8-mediated p53-independent apoptosis of human carcinoma cells.

    PubMed

    Lin, Meng-Liang; Lu, Yao-Cheng; Su, Hong-Lin; Lin, Hsin-Ting; Lee, Chuan-Chun; Kang, Shang-En; Lai, Tan-Chen; Chung, Jing-Gung; Chen, Shih-Shun

    2011-04-01

    Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.

  12. Neutrophil IL-1β processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation and is dependent on K+ efflux.

    PubMed

    Karmakar, Mausita; Katsnelson, Michael; Malak, Hesham A; Greene, Neil G; Howell, Scott J; Hise, Amy G; Camilli, Andrew; Kadioglu, Aras; Dubyak, George R; Pearlman, Eric

    2015-02-15

    Although neutrophils are the most abundant cells in acute infection and inflammation, relatively little attention has been paid to their role in inflammasome formation and IL-1β processing. In the present study, we investigated the mechanism by which neutrophils process IL-1β in response to Streptococcus pneumoniae. Using a murine model of S. pneumoniae corneal infection, we demonstrated a requirement for IL-1β in bacterial clearance, and we showed that Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 are essential for IL-1β production and bacterial killing in the cornea. Neutrophils in infected corneas had multiple specks with enzymatically active caspase-1 (YVAD-FLICA 660), and bone marrow neutrophils stimulated with heat-killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks when stained for NLRP3, ASC, or Caspase-1. High-molecular mass ASC complexes were also detected, consistent with oligomer formation. Pneumolysin induced K(+) efflux in neutrophils, and blocking K(+) efflux inhibited caspase-1 activation and IL-1β processing; however, neutrophils did not undergo pyroptosis, indicating that K(+) efflux and IL-1β processing is not a consequence of cell death. There was also no role for lysosomal destabilization or neutrophil elastase in pneumolysin-mediated IL-1β processing in neutrophils. Taken together, these findings demonstrate an essential role for neutrophil-derived IL-1β in S. pneumoniae infection, and they elucidate the role of the NLRP3 inflammasome in cleavage and secretion of IL-1β in neutrophils. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases. Copyright © 2015 by The American Association of Immunologists, Inc.

  13. ECRG4 is a negative regulator of caspase-8-mediated apoptosis in human T-leukemia cells.

    PubMed

    Matsuzaki, Junichi; Torigoe, Toshihiko; Hirohashi, Yoshihiko; Kamiguchi, Kenjiro; Tamura, Yasuaki; Tsukahara, Tomohide; Kubo, Terufumi; Takahashi, Akari; Nakazawa, Emiri; Saka, Eri; Yasuda, Kazuyo; Takahashi, Shuji; Sato, Noriyuki

    2012-05-01

    We previously established Fas-resistant variant clones from the human T-cell leukemia lines Jurkat and SUP-T13. Comparative gene expression analysis of the Fas-resistant and Fas-sensitive clones revealed several genes that were aberrantly expressed in the Fas-resistant clones. One of the genes, esophageal cancer-related gene 4 (ECRG4), contained a VDAC2-like domain that might be associated with apoptotic signals. In the present study, we examined the subcellular localization and function of ECRG4 in Fas-mediated apoptosis. By confocal fluorescence microscopy, ECRG4-EGFP fusion protein was detected in mitochondria, endoplasmic reticulum and the Golgi apparatus in gene-transfected HeLa cells. Overexpression of ECRG4 in Fas-sensitive Jurkat cells inhibited mitochondrial membrane permeability transition, leading to resistance against Fas-induced apoptosis. Tumor necrosis factor-alpha-induced apoptosis was also suppressed in ECRG4-overexpressing Jurkat cells. Immunoprecipitation assay demonstrated that ECRG4 is associated with procaspase-8. The inhibitory mechanism included the inhibition of caspase-8 activity and Bid cleavage. Since ECRG4 expression is downregulated in activated T cells, our results suggest that ECRG4 is a novel antiapoptotic gene which is involved in the negative regulation of caspase-8-mediated apoptosis in T cells.

  14. Brittle to ductile transition in cleavage fracture

    SciTech Connect

    Argon, A.S.; Berg, Q.

    1992-09-30

    The problem of interpretation of fracture transition from brittle to ductile or vice versa is the subject of study. An instrumented tapered double cantilever beam (TDCB) has been developed as a definitive tool in the study of the intrinsic mechanism in single crystalline samples. In this experiment, the crack velocity is directly proportional to actuator velocity. In experiments performed on TDCB shaped Si single crystals, oriented for cleavage on either [l brace]111[r brace] or [l brace]110[r brace] planes, a number of troubling features of jerky carck extension were encountered. Evidence suggests that nucleation of dislocation loops from crack tip is easier than moving these dislocations away from crack tip. 14 refs, 1 fig.

  15. Mn porphyrin in combination with ascorbate acts as a pro-oxidant and mediates caspase-independent cancer cell death.

    PubMed

    Evans, Myron K; Tovmasyan, Artak; Batinic-Haberle, Ines; Devi, Gayathri R

    2014-03-01

    Resistance to therapy-mediated apoptosis in inflammatory breast cancer, an aggressive and distinct subtype of breast cancer, was recently attributed to increased superoxide dismutase (SOD) expression, glutathione (GSH) content, and decreased accumulation of reactive species. In this study, we demonstrate the unique ability of two Mn(III) N-substituted pyridylporphyrin (MnP)-based SOD mimics (MnTE-2-PyP(5+) and MnTnBuOE-2-PyP(5+)) to catalyze oxidation of ascorbate, leading to the production of excessive levels of peroxide, and in turn cell death. The accumulation of peroxide, as a consequence of MnP+ascorbate treatment, was fully reversed by the administration of exogenous catalase, showing that hydrogen peroxide is essential for cell death. Cell death as a consequence of the action of MnP+ascorbate corresponded to decreases in GSH levels, prosurvival signaling (p-NF-κB, p-ERK1/2), and in expression of X-linked inhibitor of apoptosis protein, the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and annexin V staining, administration of a pan-caspase inhibitor, Q-VD-OPh, did not reverse the observed cytotoxicity. MnP+ascorbate-treated cells showed nuclear translocation of apoptosis-inducing factor, suggesting the possibility of a mechanism of caspase-independent cell death. Pharmacological ascorbate has already shown promise in recently completed phase I clinical trials, in which its oxidation and subsequent peroxide formation was catalyzed by endogenous metalloproteins. The catalysis of ascorbate oxidation by an optimized metal-based catalyst (such as MnP) carries a large therapeutic potential as an anticancer agent by itself or in combination with other modalities such as radio- and chemotherapy.

  16. Induction of caspase-mediated apoptosis and cell-cycle G1 arrest by selenium metabolite methylselenol.

    PubMed

    Wang, Zaisen; Jiang, Cheng; Lü, Junxuan

    2002-07-01

    Previous work based on mono-methyl selenium compounds that are putative precursors of methylselenol has strongly implicated this metabolite in the induction of caspase-mediated apoptosis of human prostate carcinoma and leukemia cells and G1 arrest in human vascular endothelial and cancer epithelial cells. To test the hypothesis that methylselenol itself is responsible for exerting these cellular effects, we examined the apoptotic action on DU145 human prostate cancer cells and the G1 arrest effect on the human umbilical vein endothelial cells (HUVECs) of methylselenol generated with seleno-L-methionine as a substrate for L-methionine-alpha-deamino-gamma-mercaptomethane lyase (EC4.4.1.11, also known as methioninase). Exposure of DU145 cells to methylselenol so generated in the sub-micromolar range led to caspase-mediated cleavage of poly(ADP-ribose) polymerase, nucleosomal DNA fragmentation, and morphologic apoptosis and resulted in a profile of biochemical effects similar to that of methylseleninic acid (MSeA) exposure as exemplified by the inhibition of phosphorylation of protein kinase AKT and extracellularly regulated kinases 1/2. In HUVEC, methylselenol exposure recapitulated the G1 arrest action of MSeA in mitogen-stimulated G1 progression during mid-G1 to late G1. This stage specificity was mimicked by inhibitors of phosphatidylinositol 3-kinase. The results support methylselenol as an active selenium metabolite for inducing caspase-mediated apoptosis and cell-cycle G1 arrest. This cell-free methylselenol-generation system is expected to have significant usefulness for studying the biochemical and molecular targeting mechanisms of this critical metabolite and may constitute the basis of a novel therapeutic approach for cancer, using seleno-L-methionine as a prodrug. Copyright 2002 Wiley-Liss, Inc.

  17. The CB1/VR1 agonist arvanil induces apoptosis through an FADD/caspase-8-dependent pathway

    PubMed Central

    Sancho, Rocío; de la Vega, Laureano; Appendino, Giovanni; Di Marzo, Vincenzo; Macho, Antonio; Muñoz, Eduardo

    2003-01-01

    Arvanil (N-arachidonoylvanillamine), a nonpungent capsaicin–anandamide hybrid molecule, has been shown to exert biological activities through VR1/CB1-dependent and -independent pathways. We have found that arvanil induces dose-dependent apoptosis in the lymphoid Jurkat T-cell line, but not in peripheral blood T lymphocytes. Apoptosis was assessed by DNA fragmentation through cell cycle and TUNEL analyses. Arvanil-induced apoptosis was initiated independently of any specific phase of the cell cycle, and it was inhibited by specific caspase-8 and -3 inhibitors and by the activation of protein kinase C. In addition, kinetic analysis by Western blots and fluorimetry showed that arvanil rapidly activates caspase-8, -7 and -3, and induces PARP cleavage. The arvanil-mediated apoptotic response was greatly inhibited in the Jurkat-FADDDN cell line, which constitutively expresses a negative dominant form of the adapter molecule Fas-associated death domain (FADD). This cell line does not undergo apoptosis in response to Fas (CD95) stimulation. Using a cytofluorimetric approach, we have found that arvanil induced the production of reactive oxygen species (ROS) in both Jurkat-FADD+ and Jurkat-FADDDN cell lines. However, ROS accumulation only plays a residual role in arvanil-induced apoptosis. These results demonstrate that arvanil-induced apoptosis is essentially mediated through a mechanism that is typical of type II cells, and implicates the death-inducing signalling complex and the activation of caspase-8. This arvanil-apoptotic activity is TRPV1 and CB-independent, and can be of importance for the development of potential anti-inflammatory and antitumoral drugs. PMID:14530215

  18. WP1066 disrupts Janus kinase-2 and induces caspase-dependent apoptosis in acute myelogenous leukemia cells.

    PubMed

    Ferrajoli, Alessandra; Faderl, Stefan; Van, Quin; Koch, Patricia; Harris, David; Liu, Zhiming; Hazan-Halevy, Inbal; Wang, Yongtao; Kantarjian, Hagop M; Priebe, Waldemar; Estrov, Zeev

    2007-12-01

    Several cytokines and growth factors that stimulate the proliferation of acute myelogenous leukemia (AML) cells transduce their signals by activating the transcription factor Janus-activated kinase 2 (JAK2). Accordingly, the inhibition of JAK2 or of its downstream signaling pathways suppresses the proliferation of AML cells. Because (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S0-1-phenylethyl)acrylamide) (WP1066) is a novel analogue of the JAK2 inhibitor AG490, we tested its activity in AML cells and investigated its mechanism of action. Using clonogenic assays, we found that although WP1066 had a marginal effect on normal marrow progenitors, it inhibited the proliferation of AML colony-forming cells obtained from patients with newly diagnosed AML and that of the AML cell lines OCIM2 and K562. WP1066 inhibited OCIM2 cell multiplication by inducing accumulation of cells at the G(0)-G(1) phase of the cell cycle. Similar to its parent compound AG490, WP1066 inhibited the phosphorylation of JAK2, but unlike AG490, WP1066 also degraded JAK2 protein, thereby blocking its downstream signal transducer and activator of transcription (STAT) and phosphoinositide-3-kinase pathways. These effects resulted in the activation of the caspase pathway. Incubation of both OCIM2 and K562 cells with WP1066 activated caspase-3, induced cleavage of poly(ADP-ribose) polymerase, and caused caspase-dependent apoptotic cell death. Thus, WP1066 is a potent JAK2 inhibitor whose effects in AML and other hematologic malignancies merit further investigation.

  19. Human Innate Immunity to Toxoplasma gondii Is Mediated by Host Caspase-1 and ASC and Parasite GRA15

    PubMed Central

    Gov, Lanny; Karimzadeh, Alborz; Ueno, Norikiyo; Lodoen, Melissa B.

    2013-01-01

    ABSTRACT   Interleukin-1β (IL-1β) functions as a key regulator of inflammation and innate immunity. The protozoan parasite Toxoplasma gondii actively infects human blood monocytes and induces the production of IL-1β; however, the host and parasite factors that mediate IL-1β production during T. gondii infection are poorly understood. We report that T. gondii induces IL-1β transcript, processing/cleavage, and release from infected primary human monocytes and THP-1 cells. Treating monocytes with the caspase-1 inhibitor Ac-YVAD-CMK reduced IL-1β release, suggesting a role for the inflammasome in T. gondii-induced IL-1β production. This was confirmed by performing short hairpin RNA (shRNA) knockdown of caspase-1 and of the inflammasome adaptor protein ASC. IL-1β induction required active parasite invasion of monocytes, since heat-killed or mycalolide B-treated parasites did not induce IL-1β. Among the type I, II, and III strains of T. gondii, the type II strain induced substantially more IL-1β mRNA and protein release than did the type I and III strains. Since IL-1β transcript is known to be induced downstream of NF-κB signaling, we investigated a role for the GRA15 protein, which induces sustained NF-κB signaling in a parasite strain-specific manner. By infecting human monocytes with a GRA15-knockout type II strain and a type I strain stably expressing type II GRA15, we determined that GRA15 is responsible for IL-1β induction during T. gondii infection of human monocytes. This research defines a pathway driving human innate immunity by describing a role for the classical inflammasome components caspase-1 and ASC and the parasite GRA15 protein in T. gondii-induced IL-1β production. PMID:23839215

  20. OH cleavage from tyrosine: debunking a myth

    PubMed Central

    Bury, Charles S.; Carmichael, Ian; Garman, Elspeth F

    2017-01-01

    During macromolecular X-ray crystallography experiments, protein crystals held at 100 K have been widely reported to exhibit reproducible bond scission events at doses on the order of several MGy. With the objective to mitigate the impact of radiation damage events on valid structure determination, it is essential to correctly understand the radiation chemistry mechanisms at play. OH-cleavage from tyrosine residues is regularly cited as amongst the most available damage pathways in protein crystals at 100 K, despite a lack of widespread reports of this phenomenon in protein crystal radiation damage studies. Furthermore, no clear mechanism for phenolic C—O bond cleavage in tyrosine has been reported, with the tyrosyl radical known to be relatively robust and long-lived in both aqueous solutions and the solid state. Here, the initial findings of Tyr –OH group damage in a myrosinase protein crystal have been reviewed. Consistent with that study, at increasing doses, clear electron density loss was detectable local to Tyr –OH groups. A systematic investigation performed on a range of protein crystal damage series deposited in the Protein Data Bank has established that Tyr –OH electron density loss is not generally a dominant damage pathway in protein crystals at 100 K. Full Tyr aromatic ring displacement is here proposed to account for instances of observable Tyr –OH electron density loss, with the original myrosinase data shown to be consistent with such a damage model. Systematic analysis of the effects of other environmental factors, including solvent accessibility and proximity to di­sulfide bonds or hydrogen bond interactions, is also presented. Residues in known active sites showed enhanced sensitivity to radiation-induced disordering, as has previously been reported. PMID:28009542

  1. Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties

    SciTech Connect

    Lawrence, C.P.; Chow, S.C.

    2012-11-15

    The caspase inhibitors, benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl (Cbz)-Ile-Glu (OMe)-Thr-Asp (OMe)-FMK (z-IETD-FMK) at non-toxic doses were found to be immunosuppressive and inhibit human T cell proliferation induced by mitogens and IL-2 in vitro. Both caspase inhibitors were shown to block NF-κB in activated primary T cells, but have little inhibitory effect on the secretion of IL-2 and IFN-γ during T cell activation. However, the expression of IL-2 receptor α-chain (CD25) in activated T cells was inhibited by both z-VAD-FMK and z-IETD-FMK, whereas the expression of the early activated T cell marker, CD69 was unaffected. During primary T cell activation via the antigen receptor, both caspase-8 and caspase-3 were activated and processed to their respective subunits, but neither caspase inhibitors had any effect on the processing of these two caspases. In sharp contrast both caspase inhibitors readily blocked apoptosis and the activation of caspases during FasL-induced apoptosis in activated primary T cells and Jurkat T cells. Collectively, the results demonstrate that both z-VAD-FMK and z-IETD-FMK are immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. -- Highlights: ► Caspase-8 and caspase-3 were activated during T cell activation and proliferation. ► T cell proliferation was blocked by caspase inhibitors. ► Caspase activation during T cell proliferation was not block by caspase inhibitors.

  2. Inflammatory arthritis in caspase-1 gene deficient mice: Contribution of proteinase 3 for caspase-1-independent production of bioactive IL-1β

    PubMed Central

    Joosten, Leo A.B.; Netea, Mihai G.; Fantuzzi, Giamila; Koenders, Marije I.; Helsen, Monique M.A.; Sparrer, Helmut; Pham, Christine T.; van der Meer, Jos W.M.; Dinarello, Charles A.; van den Berg, Wim B.

    2010-01-01

    Objective Caspase-1 is a known cysteine proteases and is a critical component of the inflammasome. Caspase-1 and neutrophil serine proteases, such as proteinase 3 (PR3) can process pro-IL-1β a crucial cytokine linked to the pathogenesis of rheumatoid arthritis, but their relative importance is unknown. Methods To this end we induced acute and chronic arthritis in caspase-1−/− mice and investigated the lack of caspase-1 on joint swelling, cartilage metabolism and joint pathology. In addition, caspase-1 activity was inhibited in mice lacking active cysteine proteases and evaluated the effect of dual blockade of caspase-1 and serine proteinase on arthritis severity and joint pathology. Results Surprisingly, caspase-1−/− mice developed joint swelling similar to wild-type mice in models of neutrophil-dominated arthritis. Joint fluid concentrations of bioactive IL-1β were comparable in caspase-1−/− mice and controls. In contrast, induction of chronic arthritis with minimal numbers of neutrophils in caspase-1−/− mice lead to reduced joint inflammation and cartilage damage, implying caspase-1 dependence. In mice lacking neutrophil serine PR3, inhibition caspase-1 activity results in decreased bioactive IL-1β concentrations in synovial tissue and less suppression of chondrocyte anabolic function. In addition, dual blockade of both PR-3 and caspase-1 lead to protection against cartilage and bone destruction. Conclusions We conclude that caspase-1 deficiency does not affect neutrophil-dominated joint inflammation, whereas in chronic arthritis the lack of caspase-1 results in reduced joint pathology. This study implies that caspase-1 inhibitors are not able to interfere with the whole spectrum of IL-1β production and hence may be of therapeutic value only in inflammatory conditions where limited numbers of neutrophils are present. PMID:19950280

  3. Viola plant cyclotide vigno 5 induces mitochondria-mediated apoptosis via cytochrome C release and caspases activation in cervical cancer cells.

    PubMed

    Esmaeili, Mohammad Ali; Abagheri-Mahabadi, Nazanin; Hashempour, Hossein; Farhadpour, Mohsen; Gruber, Christian W; Ghassempour, Alireza

    2016-03-01

    Cyclotides describe a unique cyclic peptide family that displays a broad range of biological activities including uterotonic, anti-bacteria, anti-cancer and anti-HIV. The vigno cyclotides consist of vigno 1-10 were reported recently from Viola ignobilis. In the present study, we examined the effects of vigno 5, a natural cyclopeptide from V. ignobilis, on cervical cancer cells and the underlying mechanisms. We found that vigno 5-treated Hela cells were killed off by apoptosis in a dose-dependent manner within 24h, and were characterized by the appearance of nuclear shrinkage, cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. The mitochondrial pathway of apoptosis revealed that cytochrome C is released from mitochondria to cytosol, associated with the activation of caspase-9 and -3, and the cleavage of poly (ADP-ribose) polymerase (PARP). Overall, the results indicate that vigno 5 induces apoptosis in part via the mitochondrial pathway, which is associated with a release of cytochrome C and elevated activity of caspase-9 and -3 in Hela cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Increased placental XIAP and caspase 3 is associated with increased placental apoptosis in a baboon model of maternal nutrient reduction (MNR)

    PubMed Central

    Arroyo, Juan A; Li, Cun; Schlabritz-Loutsevitch, Natalia; McDonald, Tom; Nathanielsz, Peter; Galan, Henry L

    2010-01-01

    Objective Our objective was to determine signaling molecules and apoptosis rate in the term placenta of a baboon model of maternal nutrient reduction (MNR). Study Design Female baboons were fed ad libitum for controls (CTR; n=7) or 70% of CTR diet (MNR; n=6) from 30 to 165 days of gestation (dG) with necropsy at 165 dG. Placental tissues were collected, fixed for immunohistochemistry or snap frozen to measure ERK, AKT, JNK, XIAP and caspase 3. Placental villous apoptosis was determined by TUNEL and cytokeratin 18 cleavage. Results Compared to CTR, MNR placentas demonstrated: reduced placental weight (p<0.02), decreased p-ERK (p<0.04), increase placental villous apoptosis (p<0.001), increased villous cytokeratin 18 cleavage, increased XIAP protein (p<0.007) and increased active caspase 3 (p<0.02). Conclusion We conclude that placental apoptosis is increased in this baboon model of MNR at term and that the increase in XIAP may be a protective mechanism against this apoptosis. PMID:20599184

  5. Suture compression induced midpalatal suture chondrocyte apoptosis with increased caspase-3, caspase-9, Bad, Bak, Bax and Bid expression.

    PubMed

    Lan, Tingting; Zhao, Hanchi; Xiang, Bilu; Wang, Jun; Liu, Yang

    2017-07-22

    Previous studies found bone resorption and chondrocytes loss in mouse models of mid-palatal suture when given continuous compressive force, although chondrocytes response remained unknown. Herein, we design this study to determine how continuous compression force induces chondrocytes apoptosis. Thirty C57BL/6 male mice (aged 6 weeks) were randomly assigned into controls (not ligated to a spring), blank controls (ligated with no compression) and the compression group (ligated with 20-g compression). After 4 d, palatal tissues were sampled and stained by TB and safranin-O. Tunel staining measured the percentage of apoptotic chondrocytes, and immunohistochemistry was performed to label apoptosis-associated proteins (e.g., Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, caspase-3, caspase-8 and caspase-9). Intergroup comparison was made by the rank sum test, and P < 0.05 was defined as statistical significance. After 7d of induction, TB and safranin-O staining revealed that the cartilage area in the compression group was significantly decreased, while the control group remained largely unaltered. Tunel staining showed that apoptotic cell numbers in the mid-palatal suture were significantly higher than the control group. Immunohistochemistry showed that mice in the compression group had significantly increased expression of caspase-3, caspase-9, Bad, Bak, Bax and Bid; However, caspase-8 remained unaltered. No expression of Bcl-2 and Bcl-xl was detected. Continuous compression force induces chondrocytes apoptosis in the mid-palatal suture. This process might be associated with the mitochondrial pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Discovery of a highly selective caspase-3 substrate for imaging live cells.

    PubMed

    Vickers, Chris J; González-Páez, Gonzalo E; Wolan, Dennis W

    2014-10-17

    Caspases are a family of cysteine proteases that are well-known for their roles in apoptosis and inflammation. Recent studies provide evidence that caspases are also integr