Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.62 (caspase-9)
 7,507 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intradermal vaccination by gene gun efficiently delivers DNA vaccines into DCs of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. DCs, however, have a limited life span, hindering their long-term ability to prime antigen-specific T cells. We reason that a strategy that prolongs the survival of DNA-transduced DCs will enhance priming of antigen-specific T cells and DNA vaccine potency. Here we show that codelivery of DNA encoding inhibitors of apoptosis (BCL-xL, BCL-2, XIAP, dominant negative caspase-9, or dominant negative caspase-8) with DNA encoding model antigens prolongs the survival of transduced DCs. More importantly, vaccinated mice exhibited significant enhancement in antigen-specific CD8+ T cell immune responses, resulting in a potent antitumor effect against antigen-expressing tumors. Among these antiapoptotic factors, BCL-xL demonstrated the greatest enhancement in antigen-specific immune responses and antitumor effects. Thus, coadministration of DNA vaccines with DNA encoding antiapoptotic proteins represents an innovative approach to enhance DNA vaccine potency.
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PMID:Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins. 1284 54

Procaspase-3 (p32) is processed by upstream caspases to p12 and p20 subunits, which heterodimerize. Concomitant with formation of the active heterotetramer, p20 is autoprocessed to p17. Treatment of HL-60 cells with lactacystin, a selective inhibitor of the proteasome, exponentially increased caspase-3-like hydrolytic activity and induced apoptosis but had little or no effect on the activity of upstream caspase-8, caspase-9, or granzyme B. Lactacystin treatment decreased the p32 zymogen and evoked the accumulation of the p17 and p12 subunits. Treatment of transfected human retinoblast 911 cells with a proteasome inhibitor evoked the accumulation of epitope-tagged p12, p17, and p20 but had no effect on p32 zymogen. This result suggests that caspase-3 subunits, in contrast to the zymogen, are unstable because of degradation by the ubiquitin-proteasome system. Ubiquitin conjugates of p12 and p17 accumulated in cells that were cotransfected with p12 and a caspase inactive mutant of p17. Substitution of arginine for all eight lysines of p12 almost abolished its ubiquitination. Any single lysine or lysine pair was sufficient for p12 ubiquitination. Lactacystin treatment of HL-60 cells induced proteolytic processing of the X-linked inhibitor of apoptosis (XIAP) and decreased full-length XIAP, which is known to have ubiquitin-protein ligase activity for active caspase-3. These findings indicate that caspase-3 subunits can be degraded by the ubiquitin-proteasome system and suggest that lactacystin induces apoptosis in part by disabling the ubiquitin-protein ligase function of XIAP and by stabilizing active caspase-3 subunits.
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PMID:Preservation of caspase-3 subunits from degradation contributes to apoptosis evoked by lactacystin: any single lysine or lysine pair of the small subunit is sufficient for ubiquitination. 1286 38

Interactions between pharmacologic NF-kappaB inhibitors (eg, Bay 11-7082, SN-50) and the checkpoint abrogator UCN-01 have been examined in human multiple myeloma (MM) cells. Exposure of U266 cells to Bay 11-7082 (Bay) in combination with UCN-01 resulted in the abrogation of NF-kappaB/DNA binding activity and the synergistic induction of apoptosis. Comparable synergism was observed in other MM cell lines and patient-derived CD138+ cells and between an inhibitory peptide of NF-kappaB (SN50) and UCN-01. Bay/UCN-01-mediated lethality involved mitochondrial dysfunction, caspase cleavage, and poly adenosine diphosphate-ribose polymerase (PARP) degradation. Although Bay modestly blocked UCN-01-induced extracellular signal-regulated kinase (ERK) phosphorylation, coadministration activated c-Jun N-terminal kinase (JNK) and cdc2/cdk1 and down-regulated Mcl-1, XIAP, and Bcl-xL. Transfection with a constitutively activated mitogen-activated protein kinase kinase (MEK1)/green fluorescent protein (GFP) construct failed to block apoptosis induced by Bay/UCN-01 but significantly attenuated MEK inhibitor (U0126)/UCN-01-induced lethality. Inhibiting JNK activation with SP600125 or D-JNKI1 peptide markedly reduced Bay/UCN-01-mediated mitochondrial dysfunction and apoptosis and the down-regulation of Mcl-1, XIAP, and Bcl-xL but not of cdc2/cdk1 activation. Stable transfection of cells with dominant-negative caspase-9 dramatically diminished Bay/UCN-01 lethality without altering JNK or cdc2/cdk1 activation. Neither interleukin-6 (IL-6)- nor fibronectin-mediated adherence conferred resistance to Bay/UCN-01-induced apoptosis. Together, these findings suggest that a strategy combining UCN-01 with disruption of the IkappaB kinase (IKK)/IkappaB/NF-kappaB pathway warrants attention in MM.
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PMID:Interruption of the NF-kappaB pathway by Bay 11-7082 promotes UCN-01-mediated mitochondrial dysfunction and apoptosis in human multiple myeloma cells. 1464 3

Apoptosis occurs in the placenta throughout gestation, with a greater frequency near term in comparison to the first trimester. The Fas/FasL system represents one of the main apoptotic pathways controlling placental apoptosis. Although first trimester trophoblast cells express both Fas and FasL, they are resistant to Fas-induced apoptosis. Therefore, trophoblast resistance to Fas-mediated apoptosis may be due to the inhibition of the pathway downstream of Fas stimulation. Expression levels of X-linked inhibitor of apoptosis (XIAP) were recently shown to decrease in third trimester placentas, correlating with an increase in placental apoptosis. As a potent caspase inhibitor, XIAP prevents the activation of caspase-9 through its BIR3 domain and caspase-3 activation via the linker-BIR2 domain. In the present study, high levels of the active form of XIAP were detected in first trimester trophoblast cells, whereas term placental tissue samples predominantly expressed the inactive form of XIAP. Using a XIAP inhibitor, phenoxodiol, we demonstrate that XIAP inactivation sensitizes trophoblast cells to Fas stimulation, as evidenced by the anti-Fas mAb-induced decrease in trophoblast cell viability and increase in caspase-8, caspase-9 and caspase-3 activation. This suggests a functional role for XIAP in the regulation of the Fas apoptotic cascade in trophoblast cells during pregnancy.
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PMID:X-linked inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-mediated apoptosis. 1466 4

Many environmental and therapeutic agents initiate apoptotic cell death by inducing the release of cytochrome c from the mitochondria, which activates Apaf-1 (apoptotic protease-activating factor-1). This large (approximately 130kD) protein is a mammalian homologue of CED-4, an essential protein involved in programmed cell death in the nematode C. elegans. Cytochrome c activates Apaf-1, which oligomerizes to form an approximately 700-1400-kDa caspase-activating complex known as the Apaf-1 apoptosome. Caspase-9, an initiator caspase, is then recruited to the complex by binding to Apaf-1 through CARD-CARD (caspase recruitment domain) interactions to form a holoenzyme complex. Subsequently, the Apaf-1/caspase-9 holoenzyme complex recruits the effector caspase-3 via an interaction between the active site cysteine in caspase-9 and the critical aspartate, which is the cleavage site for generating the large and small subunits of caspase-3 that constitute the activated form of caspase-3. This initiates the caspase cascade that is responsible for the execution phase of apoptosis. Intracellular levels of K+, XIAP an inhibitor of apoptosis protein, and at least two mitochondrial released proteins, Smac/DIABLO and Omi/Htra 2 a serine protease, tightly regulate formation and function of the apoptosome. Thus, a number of physiological mechanisms ensure that the apoptosome complex is only fully assembled and functional when the cell is irrevocably committed to die. It is interesting that more recent studies show that a variety of small molecules can directly activate or inhibit caspase activation by interfering with the formation and function of the apoptosome complex. The cytotoxicity of many conventional chemotherapeutic drugs rests on their ability to induce apoptosome formation and apoptosis. Defects in this pathway can result in drug resistance, and the discovery that small molecules can directly activate or inhibit the apoptosome may provide new alternative treatments for cancer.
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PMID:Chemical-induced apoptosis: formation of the Apaf-1 apoptosome. 1470 65

In this issue of Cancer Cell, Schimmer et al. report the identification of small molecule antagonists of XIAP that overcome its inhibition of caspase-3. It was remarkable that the compounds directly induced cell death in tumor cells while having little toxicity on normal cells. This suggests that caspases are already activated in tumor cells, which is different from the caspase activation status in normal mammalian cells. In comparison with Smac peptides targeting XIAP-mediated caspase-9 inhibition, which do not directly induce cell death, it appears that liberating downstream caspases rather than upstream caspases may be a preferred strategy for cancer drug discovery.
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PMID:Antagonizing XIAP-mediated caspase-3 inhibition. Achilles' heel of cancers? 1474 24

The apoptosome is a large caspase-activating ( approximately 700-1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is approximately 135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9(1-130)) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9(1-130), demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6-8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.
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PMID:Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex. 1499 23

A functional disorder associated with weightlessness is well documented in osteoblasts. The apototic features of this disorder are poorly understood. Harmful stress induces apoptosis in cells via mitochondria and/or Fas. The Bax triggers cytochrome c release from mitochondria, which can be blocked by the Bcl-2. Released cytochrome c then activates the initiator caspase, caspase-9, which can be blocked by the anti-apototic (IAP) family of molecules. The effector caspase, caspase-3, finally exerts DNA fragmentation. We conducted this study to examine the apoptotic effects of vector-averaged gravity on normal human osteoblastic cells. Cell culture flasks were incubated on the clinostat, which generated vector-averaged gravity condition (simulated microgravity) for 12, 24, 48, and 96 hours. Upon termination of clinostat cultures, the cell number and cell viability were assessed. DNA fragmentation was analyzed on the agarose-gel electrophoresis. The mRNA levels for Bax, Bcl-2, XIAP, and caspase-3 genes were analyzed by semi-quantitative RT-PCR. Twenty-four hours after starting clinostat rotation, the ratios of Bax/Bcl-2 mRNA levels (indicator of apoptosis) were significantly increased to 136% of the 1G static controls. However, the XIAP mRNA levels (anti-apoptotic molecule) were increased concomitantly to 138% of the 1G static controls. Thus, cell proliferation or cell viability was not affected by vector-averaged gravity. DNA fragmentation was not observed in clinostat group as well as in control group. Finally, the caspase-3 mRNA levels were not affected by vector-averaged gravity. Simulated microgravity might modulate some apoptotic signals upstream the mitochondrial pathway.
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PMID:Antagonism between apoptotic (Bax/Bcl-2) and anti-apoptotic (IAP) signals in human osteoblastic cells under vector-averaged gravity condition. 1503 9

Present studies demonstrate that treatment with the histone deacetylases inhibitor LAQ824, a cinnamic acid hydroxamate, increased the acetylation of histones H3 and H4, as well as induced p21(WAF1) in the human T-cell acute leukemia Jurkat, B lymphoblast SKW 6.4, and acute myelogenous leukemia HL-60 cells. This was associated with increased accumulation of the cells in the G(1) phase of the cell cycle, as well as accompanied by the processing and activity of caspase-9 and -3, and apoptosis. Exposure to LAQ824 increased the mRNA and protein expressions of the death receptors DR5 and/or DR4, but reduced the mRNA and protein levels of cellular FLICE-inhibitory protein (c-FLIP). As compared with treatment with Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or LAQ824 alone, pretreatment with LAQ824 increased the assembly of Fas-associated death domain and caspase-8, but not of c-FLIP, into the Apo-2L/TRAIL-induced death-inducing signaling complex. This increased the processing of caspase-8 and Bcl-2 interacting domain (BID), augmented cytosolic accumulation of the prodeath molecules cytochrome-c, Smac and Omi, as well as led to increased activity of caspase-3 and apoptosis. Treatment with LAQ824 also down-regulated the levels of Bcl-2, Bcl-x(L), XIAP, and survivin. Partial inhibition of apoptosis due to LAQ824 or Apo-2L/TRAIL exerted by Bcl-2 overexpression was reversed by cotreatment with LAQ824 and Apo-2L/TRAIL. Significantly, cotreatment with LAQ824 increased Apo-2L/TRAIL-induced apoptosis of primary acute myelogenous leukemia blast samples isolated from 10 patients with acute myelogenous leukemia. Taken together, these findings indicate that LAQ824 may have promising activity in augmenting Apo-2L/TRAIL-induced death-inducing signaling complex and apoptosis of human acute leukemia cells.
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PMID:Cotreatment with histone deacetylase inhibitor LAQ824 enhances Apo-2L/tumor necrosis factor-related apoptosis inducing ligand-induced death inducing signaling complex activity and apoptosis of human acute leukemia cells. 1505 15

The Apaf-1 apoptosome is a multi-subunit caspase-activating scaffold that is assembled in response to diverse forms of cellular stress that culminate in apoptosis. To date, most studies on apoptosome composition and function have used apoptosomes reassembled from recombinant or purified proteins. Thus, the precise composition of native apoptosomes remains unresolved. Here, we have used a one-step immunopurification approach to isolate catalytically active Apaf-1/caspase-9 apoptosomes, and have identified the major constituents of these complexes using mass spectrometry methods. Using this approach, we have also assessed the ability of putative apoptosome regulatory proteins, such as Smac/DIABLO and PHAPI, to regulate the activity of native apoptosomes. We show that Apaf-1, caspase-9, caspase-3 and XIAP are the major constituents of native apoptosomes and that cytochrome c is not stably associated with the active complex. We also demonstrate that the IAP-neutralizing protein Smac/DIABLO and the tumor-suppressor protein PHAPI can enhance the catalytic activity of apoptosome complexes in distinct ways. Surprisingly, PHAPI also enhanced the activity of purified caspase-3, suggesting that it may act as a co-factor for this protease.
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PMID:Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes. 1510 27


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