• Title, Summary, Keyword: caspase

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A Correlative Study on Aβ and CD95 Pathway Independent to Ca2+ Dependent Protease and Activation of Caspase Activation

  • Tuyet, Pham Thi Dieu
    • Journal of the Chosun Natural Science
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    • v.7 no.1
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    • pp.25-38
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    • 2014
  • Amyloid-${\beta}$-peptide ($A{\beta}$) is important in the pathogenesis of Alzheimer's disease (AD). Calpain ($Ca^{2+}$-dependent protease) and caspase-8 (the initiating caspase for the extrinsic, receptor-mediated apoptosis pathway) have been implicated in $AD/A{\beta}$ toxicity. We found that $A{\beta}$ promoted degradation of calpastatin (the specific endogenous calpain inhibitor); calpastatin degradation was prevented by inhibitors of either calpain or caspase-8. The results implied a cross-talk between the two proteases and suggested that one protease was responsible for the activity of the other one. In neuron-like differentiated PC12 cells, calpain promotes active caspase-8 formation from procaspase-8 via the $A{\beta}$ and CD95 pathways, along with degradation of the procaspase-8 processing inhibitor caspase-8 (FLICE)-like inhibitory protein, short isoform (FLIPS). Inhibition of calpain (by pharmacological inhibitors and by overexpression of calpastatin) prevents the cleavage of procaspase-8 to mature, active caspase-8, and inhibits FLIPS degradation in the $A{\beta}$-treated and CD95-triggered cells. Increased cellular Ca2+ per se results in calpain activation but does not lead to caspase-8 activation or FLIPS degradation. The results suggest that procaspase-8 and FLIPS association with cell membrane receptor complexes is required for calpain-induced caspase-8 activation. The results presented here add to the understanding of the roles of calpain, caspase- 8, and CD95 pathway in $AD/A{\beta}$ toxicity. Calpain-promoted activation of caspase-8 may have implications for other types of CD95-induced cell damage, and for nonapoptotic functions of caspase-8. Inhibition of calpain may be useful for modulating certain caspase-8-dependent processes.

Effect of Hypoxia on the Signal Transduction of Apoptosis in Osteoblasts (저산소 상태에서 조골세포 고사의 신호전달 기전)

  • Park, Young-Joo;Oh, Soh-Taek;Kang, Kyung-Hwa;Kim, Sang-Cheol
    • The korean journal of orthodontics
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    • v.33 no.6
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    • pp.453-463
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    • 2003
  • Mammalian cell is critically dependent on a continuous supply of oxygen. Even brief periods of oxygen deprivation can result in profound cellular damage. The aim of this study was to examine the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts. MC3T3El osteoblasts under hypoxic conditions ($2\%$ oxygen) resulted in apoptosis in a time-dependent manner, determined by DNA fragmentation assay and nuclear morphology, stained with fluorescent dye (Hoechst 33258) Pretreatment with Z-VAD-FMK, a pancaspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, suppressed the DNA ladder in response to hypoxia in a concentration dependent manner. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-l activity (YVADase) was detected. To confirm what caspases were involved in apoptosis, western blot analysis was performed using an anticaspase-3 or 6 antibody. The 17-kDa protein, that corresponds to the active products of caspase-3 and the 20-kDa protein of the active protein of caspase-6 were generated in hypoxia-challenged lysates, in which the full length forms of caspase-3 and 6 were evident. With a time course similar to caspase-3 and 6 activation, hypoxic stress also caused the cleavage of Lamin A, typical of caspase-6 activity. In addition, the hypoxic stress elicited the release of cytochrome c into the cytosol during apoptosis. These findings suggested that the activation of caspases accompanied by a cytochrome c release in response to hypoxia was involved in apoptotic cell death in MC3T3E1 osteoblasts.

EphA Receptors Form a Complex with Caspase-8 to Induce Apoptotic Cell Death

  • Lee, Haeryung;Park, Sunjung;Kang, Young-Sook;Park, Soochul
    • Molecules and Cells
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    • v.38 no.4
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    • pp.349-355
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    • 2015
  • EphA7 has been implicated in the regulation of apoptotic cell death in neural epithelial cells. In this report, we provide evidence that EphA7 interacts with caspase-8 to induce apoptotic cell signaling. First, a pull-down assay using biotinylated ephrinA5-Fc showed that EphA7 co-precipitated with wild type caspase-8 or catalytically inactive caspase-8 mutant. Second, co-transfection of EphA7 with caspase-8 significantly increased the number of cleaved caspase-3 positive apoptotic cells under an experimental condition where transfection of EphA7 or caspase-8 alone did not affect cell viability or apoptosis. EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not. Third, caspase-8 catalytic activity was essential for the apoptotic signaling cascade, whereas tyrosine kinase activity of the EphA4 receptor was not. Interestingly, we found that kinase-inactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable. Finally, we observed that the extracellular region of the EphA7 receptor was critical for interacting with caspase-8, whereas the cytoplasmic region of EphA7 was not. Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptor-like protein acts as a biochemical linker between the Eph receptor and caspase-8.

Expression of the Pro-Domain-Deleted Active Form of Caspase-6 in Escherichia coli

  • Lee, Phil Young;Cho, Jin Hwa;Chi, Seung Wook;Bae, Kwang-Hee;Cho, Sayeon;Park, Byoung Chul;Kim, Jeong-Hoon;Park, Sung Goo
    • Journal of Microbiology and Biotechnology
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    • v.24 no.5
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    • pp.719-723
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    • 2014
  • Caspases are a family of cysteine proteases that play an important role in the apoptotic pathway. Caspase-6 is an apoptosis effector that cleaves a variety of cellular substrates. The active form of the enzyme is required for use in research. However, it has been difficult to obtain sufficient quantities of active caspase-6 from Escherichia coli. In the present study, we constructed a caspase-6 with a 23-amino-acid deletion in the pro-domain. This engineered enzyme was expressed as a soluble protein in E. coli and was purified using affinity resin. In vitro enzyme assay and cleavage analysis revealed that the engineered active caspase-6 protein had characteristics similar to those of wild-type caspase-6. This novel method can be a valuable tool for obtaining active caspase-6 that can be used for screening caspase-6-specific substrates, which in turn can be used to elucidate the function of caspase-6 in apoptosis.

High-level Expression of Human Procaspase-9 in Escherichia coli and Purification of its GST-tagged Recombinant Protein (대장균을 이용한 세포사멸 유도 단백질 caspase-9의 발현에 관한 연구)

  • Seong, Yeong-Mo;Han, Cheol;Choe, Ju-Yeon;Park, Hyo-Jin;Seong, Geun-Hye;Nam, Min-Gyeong;Kim, Sang-Su;Kim, In-Gyeong;Gang, Seong-Man;Im, Hyang-Suk
    • Korean Journal of Microbiology
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    • v.39 no.4
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    • pp.216-222
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    • 2003
  • Human caspase-9, an essential apoptosis initiator protease, was excessively degraded when expressed in Escherichia coli under the conventional induction condition. To optimize the conditions for induction and develop a rapid purification method for obtaining significant amounts of wild-type procaspase-9, we expressed procaspase-9 as GST fusion in E. coli. The addition of 0.01 mM IPTG as an inducer to the bacterial culture and decreasing the culture temperature to 25oC improved the production of procasapse-9 protein by circumventing proteolytic degradation in E. coli. The wild-type procaspae-9 was purified to approximately 70% purity with relatively high yields using the method developed in this study. In addition, we found that GST-caspase-9 is autocatalytically cleaved after aspartic acid 315, which is the same site for processing in mammalian cells, during expression in E. coli.

Caspase-11 Promoter-GFP Construct as a Dual Reporter of Cytotoxicity and Inflammation

  • Shin, Ki-Soon;Kang, Shin-Jung
    • Animal cells and systems
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    • v.10 no.2
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    • pp.73-77
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    • 2006
  • Caspase-11 has been known as a dual regulator of apoptosis and inflammatory response. An unusual feature of caspase-11 is that its expression is induced by apoptotic or proinflammatory stimuli. Utilizing these unusual features of caspase-11, we have developed a simple and sensitive assay method to screen pro- or anti-apoptotic/inflammatory molecules. To develop this assay method, we generated a reporter construct where GFP expression is regulated by caspase-11 promoter. When several types of cultured cells were transfected with this reporter construct and subsequently treated with various apoptotic or proinflammatory molecules, expression of GFP by the activation of caspase-11 promoter was easily detected by fluorescence microscopy or spectrofluorometry. In addition, a reduction of the GFP fluorescence was detected when an agent reported to suppress caspase-11 induction was applied. These results suggest that our reporter system can be used to screen pro- or anti-apoptotic/inflammatory molecules.

Ginsenoside Rh2 Induces Apoptosis via Activation of Caspase-1 and -3 and Up-Regulation of Bax in Human Neuroblastoma

  • Kim, Young-Soak;Jin, Sung-Ha
    • Archives of Pharmacal Research
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    • v.27 no.8
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    • pp.834-839
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    • 2004
  • In human neuroblastoma SK-N-BE(2) cells undergoing apoptotic death induced by ginsenos-ide Rh2, a dammarane glycoside that was isolated from Panax ginseng C. A. Meyer, caspase-1 and caspase-3 were activated. The expression of Bax was increased in the cells treated with ginsenoside Rh2, whereas Bcl-2 expression was not altered. Treatment with caspase-1 inhibi-tor, Ac-YVAD-CMK, or caspase-3 inhibitor, Z-DEVD-FMK, partially inhibited ginsenoside Rh2-induced cell death but almost suppressed the cleavage of the 116 kDa PARP into a 85 kDa fragment. When the levels of p53 were examined in this process, p53 accumulated rapidly in the cells treated early with ginsenoside Rh2. These results suggest that activation of caspase-1 and -3 and the up-regulation of Bax are required in order for apoptotic death of SK-N-BE(2) cells to be induced by ginsenoside Rh2, and p53 plays an important role in the pathways to promote apoptosis.

Induction of Caspase-3 Dependent Apoptosis in Human Ovarian Cancer SK-OV-3 Cells by Genistein

  • Choi, Eun-Jeong;Kim, Tae-Hee;Kim, Gun-Hee;Chee, Kew-Mahn
    • Food Science and Biotechnology
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    • v.17 no.1
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    • pp.216-218
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    • 2008
  • The present study was designed to determine how the phytochemical genistein activates caspase-3 to cause cell cycle arrest and apoptosis. When human ovarian cancer SK-OV-3 cells were treated with $200\;{\mu}M$ genistein for 24 hr, cell growth decreased significantly (p<0.05). Conversely, genistein treatment significantly increased cytotoxicity (measured as lactate dehydrogenase release) under the same conditions (p<0.05). To elucidate the mechanism behind the induction of apoptosis by genistein, we studied the cell cycle and caspase-3 activation. When cells were treated with genistein, the population of cells in sub-G1 phase increased by 44.2% compared to untreated cells. Genistein caused decrease in precursor caspase-3, increase in cleaved caspase-3 and a significant increase in caspase-3 activity (p<0.05). Therefore, genistein may induce apoptosis via caspase-3 activation. However, high-dose genistein treatment must be viewed with caution because of its potential cytotoxicity.

Apoptotic Signaling Pathway by Cadmium in Hepalclc7 cells (Hepa1c1c7 세포에서 카드뮴에 의한 세포사멸 신호전달체계에 관한 연구)

  • 오경재;염정호
    • Toxicological Research
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    • v.17 no.3
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    • pp.215-223
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    • 2001
  • Cadmium is an ubiquitous toxic metal and chronic exposure to cadmium results in the accumulation of cadmium in the liver and kidneys. In contrast, acute exposure leads to damage mainly in the liver. Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. However, the molecular mechanism of cadmium-induced apoptosis is not clear in hepatocyte. To investigate the induction of apoptosis in the hepatocyte, we used mouse hepatoma cell line, Hepalclc7 cells, and analysed the molecules that involved in cadmium-induced apoptosis. Cadmium induced the genomic DNA fragmentation, PARP cleavage, and activation of caspase-3 like protease. Caspase-9 cysteine protease was activated in a time-dependent manner but caspase-8 cysteine protease was not significantly activated in cadmium-treated Hepalclc7 cells. Cadmium also induced mitochondrial dysfunction including cytochrome c release from mitochondria, change oj mitochondrial membrane potential tranition, and tranlocation of Bax Protein into mitochondria. These results strong1y indicated that the signal Pathway of apoptotic death in cadmium-treated Hepalclc7 cells is modulated by caspase cascade via mitochondria.

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Identification of Novel Binding Partners for Caspase-6 Using a Proteomic Approach

  • Jung, Ju Yeon;Lee, Su Rim;Kim, Sunhong;Chi, Seung Wook;Bae, Kwang-Hee;Park, Byoung Chul;Kim, Jeong-Hoon;Park, Sung Goo
    • Journal of Microbiology and Biotechnology
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    • v.24 no.5
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    • pp.714-718
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    • 2014
  • Apoptosis is the process of programmed cell death executed by specific proteases, the caspases, which mediate the cleavage of various vital proteins. Elucidating the consequences of this endoproteolytic cleavage is crucial to understanding cell death and other related biological processes. Although a number of possible roles for caspase-6 have been proposed, the identities and functions of proteins that interact with caspase-6 remain uncertain. In this study, we established a cell line expressing tandem affinity purification (TAP)-tagged caspase- 6 and then used LC-MS/MS proteomic analysis to analyze the caspase-6 interactome. Eight candidate caspase-6-interacting proteins were identified. Of these, five proteins (hnRNP-M, DHX38, ASPP2, MTA2, and UACA) were subsequently examined by co-immunoprecipitation for interactions with caspase-6. Thus, we identified two novel members of the caspase-6 interactome: hnRNP-M and MTA2.