Caspase-mediated cleavage of JNK during stress-induced apoptosis

The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). The JNKs are encoded by three separate genes (jnk1, jnk2, and jnk3), which are spliced alternatively to create 10 JNK isoforms that are either p46 or p54 in size. In this study, we found that the p52 form of JNK emerged in human leukemia MOLT-4 or U937 cells following X-irradiation or heat treatment. The accumulation of p52 coincided with the reduction of p54 JNK. On the other hand, the amounts of p46 JNK did not change by X-irradiation. Induction of the p52 form of JNK also paralleled the appearance of the active form of caspase-3 and was suppressed by a caspase-specific inhibitor, Ac-DEVD-CHO, but not by Ac-YVAD-CHO. In vitro cleavage assays indicated that recombinant human JNK1beta2 and JNK2beta2 were cleaved by caspase-3, and that the mutation of aspartic acid at position 413 of JNK1beta2 or 410 of JNK2beta2 to alanine abolished the cleavage. Altogether, our results demonstrated that p54 JNKs, at least JNK1beta2 and JNK2beta2, were new selective targets of caspases in JNK splicing variants, and suggested that the p52 form could serve as a marker of apoptosis.     

FANCD2 is a target for caspase 3 during DNA damage-induced apoptosis

The Fanconi anemia (FA) pathway, of which the FANCD2 protein is a key component, plays crucial roles in the maintenance of hematopoietic stem cells and suppression of carcinogenesis. However, the function of FANCD2 remains unclear. Here, we report that FANCD2 is a novel and specific substrate of caspase 3. Cleavage of FANCD2 by caspase 3 did not require either the FA core complex or mono-ubiquitylation of FANCD2, and was stimulated by p53. In addition, we identified the cleavage sites and generated cell lines that stably express a caspase-resistant FANCD2 mutant. Our data suggest that FANCD2 is regulated by caspase-mediated degradation during apoptosis induced by DNA damage.     

Deregulation of Cdc2 kinase induces caspase-3 activation and apoptosis

Progression of the cell cycle and control of apoptosis are tightly linked processes. It has been reported that manifestation of apoptosis requires cdc2 kinase activity yet the mechanism(s) of which is largely unclear. In an attempt to study the role of human MDM2 (HDM2) in interphase and mitosis, we employed the Xenopus cell-free system to study HDM2 protein stability. Interestingly, HDM2 is specifically cleaved in Xenopus mitotic extracts but not in the interphase extracts. We demonstrate that HDM2 cleavage is dependent on caspase-3 and that activation of cdc2 kinase results in caspase-3 activation in the Xenopus cell-free system. Furthermore, expression of cdc2 kinase in mammalian cells leads to activation of caspase-3 and apoptosis. Taken together, these data indicate that deregulation of cdc2 kinase activity can trigger apoptotic machinery that leads to caspase-3 activation and apoptosis.     

Sir2基因家族的功能和作用机制

Sir2(silenceinformationregulator)基因家族是一种保守的从古细菌到哺乳动物都存在的NAD 依赖的组蛋白/非组蛋白去乙酰化酶。在酵母中,Sir2连同与它相互作用的几个蛋白质在基因沉默、基因组稳定性、细胞寿命以及代谢调节上起着不可缺少的作用。其主要的作用机制是:热量限制降低了抑制物烟酰胺的浓度,从而激活了Sir2的组蛋白去乙酰化功能。在哺乳动物中,有7个Sir2同源基因,分别命名为SIRT1到SIRT7。其中SIRT1研究的最多,它在DNA损伤修复、细胞周期控制、抑制细胞凋亡、抵抗氧化逆境和延长细胞寿命方面起着重要作用。它的这些功能是通过和p53、FOXO3、Ku70和PGC-1α等蛋白质之间的相互作用而实现的。     

Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth

Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs.     

Human tissue factor pathway inhibitor-2 induces caspase-mediated apoptosis in a human fibrosarcoma cell line

Human TFPI-2 is an extracellular matrix-associated Kunitz-type serine proteinase inhibitor. We previously demonstrated that a human fibrosarcoma cell line, HT-1080, does not express TFPI-2, but genetic restoration of TFPI-2 expression in these cells markedly inhibited their growth and metastasis in vivo. In the present study, either full-length recombinant TFPI-2, or its mutated first Kunitz-type domain (R24K KD1), were offered to HT-1080 cells, and the degree of apoptosis assessed by nuclear fragmentation, ethidium bromide and acridine orange staining, fluorescence activated cell sorting, immunoblotting and gene expression profiling. R24K KD1 induced apoptosis in 69% of HT-1080 cells in a 48 h period compared to 39% for TFPI-2, while a KD1 preparation lacking a reactive site arginine/lysine residue (R24Q KD1) produced only an 18% apoptosis rate, suggesting that the observed apoptosis was related to proteinase inhibition. Immunoblotting experiments indicated increased caspase 3 and 9 activation, up-regulation of pro-apoptotic Bax and suppression of anti-apoptotic Bcl-2 protein. Finally, microarray analyses of R24K KD1-treated cells indicated elevated expression of several pro-apoptotic genes and under-expression of anti-apoptotic genes. Collectively, our results demonstrate that treatment of HT-1080 cells exogenously with either TFPI-2 or R24K KD1 activates caspase-mediated, pro-apoptotic signaling pathways resulting in apoptosis.     

Caspase-mediated cleavage of the signal-transducing IL-6 receptor subunit gp130

The present study characterizes the molecular mechanisms of CD95L-induced inhibition of IL-6 signaling, which is known to mediate hepatoprotective effects in response to various toxins. CD95L-induced caspase activation leads to degradation of gp130, thereby suppressing IL-6-induced phosphorylation of STAT3 (Tyr⁷⁰⁵) and of tyrosine phosphatase SHP2 (Tyr⁵⁸⁰). Degradation of gp130 protein in response to CD95L was largely prevented after inhibition of caspase 3 or 8. Introduction of a point mutation into a newly identified caspase cleavage site located within position 800–806 (DHVDGGD) of the cytoplasmic tail of gp130 leads to cleavage resistance of the respective receptor in an in vitro assay with recombinant active caspase 3. Correspondingly, the release of a C-terminal gp130-cleavage product of approximately 18 kDa was also inhibited after mutagenesis of this cleavage motif. In conclusion, this study demonstrates that caspase activation by CD95L antagonizes IL-6 signaling by a caspase-mediated cleavage of gp130 thereby probably counteracting hepatoprotective effects of IL-6.     

Cdk2 activity is associated with depolarization of mitochondrial membrane potential during apoptosis

In this study we show that panaxadiol, a ginseng saponin with a dammarane skeleton, induces apoptotic cell death by depolarization of mitochondrial membrane potential in human hepatoma SK-HEP-1 cells. Sequential activation of caspases-9, -3, and -7, but not of caspase-8, occurs after mitochondrial membrane depolarization and cytochrome c release from the mitochondria of panaxadiol-treated cells. Moreover, Cdk2 kinase activity, but not Cdc2 kinase activity, is markedly upregulated in the early stages of apoptosis. Olomoucine or roscovitine, specific Cdks inhibitors, effectively prevent mitochondrial membrane depolarization as well as apoptotic cell death in panaxadiol-treated cells. Thus, panaxadiol-treatment induces cell death-dependent activation of Cdk2 kinase activity, which is functionally associated with depolarization of mitochondrial membrane potential and subsequent cytochrome c release.     

Bcl-2 inhibitor and apigenin worked synergistically in human malignant neuroblastoma cell lines and increased apoptosis with activation of extrinsic and intrinsic pathways

Neuroblastoma is the most common extracranial solid tumor in infants and young children. Current treatments are not always effective and new therapies are needed. We examined efficacy of combination of the small molecule Bcl-2 inhibitor HA14-1 (HA) and the dietary isoflavonoid apigenin (APG) in human malignant neuroblastoma cells. Dose-response studies indicated that treatment with HA and APG for 24 h synergistically reduced cell viability in human malignant neuroblastoma SK-N-DZ, SH-SY5Y, and IMR32 cells. For further studies, we selected SK-N-DZ cells that showed the highest sensitivity following treatment with 2.5 μM HA, 100 μM APG, or combination (2.5 μM HA + 100 μM APG). Wright staining showed increase in morphological features of apoptosis. Cell cycle distribution and Annexin V assay showed that combination therapy caused more apoptosis than either treatment alone. Western blotting revealed that combination therapy downregulated angiogenic factors and also induced extrinsic pathway of apoptosis with activation of caspase-8 for Bid cleavage to tBid. Alterations in Bax and Bcl-2 levels resulted in an increase in Bax:Bcl-2 ratio to activate intrinsic pathway of apoptosis with mitochondrial release of cytochrome c into the cytosol and activation of proteases. Increases in calpain and caspase-3 activities generated 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Results showed that combination of HA and APG could be used for downregulation of angiogenic factors and activation of extrinsic and intrinsic pathways of apoptosis in malignant neuroblastoma cells.     

Conjugated linoleic acid induces apoptosis of murine mammary tumor cells via Bcl-2 loss

Conjugated linoleic acid (CLA) is a powerful anticancer agent in a number of tumor model systems; however, its precise mechanism of action remains elusive. Here, we report that t10,c12 CLA, a component of synthetic CLA supplements, induced apoptosis and G1 arrest of p53 mutant TM4t murine mammary tumor cells. Furthermore, t10,c12-CLA induced a time- and concentration-dependent cleavage of caspases-3 and -9, and release of cytochrome c from mitochondria to cytosol. Levels of Bcl-2 protein were decreased both in total cellular lysates and in mitochondria after t10,c12-CLA treatment; however, there was no significant change in Bax or Bak. Overexpression of Bcl-2 attenuated apoptosis in response to t10,c12-CLA treatment. These results demonstrate that t10,c12-CLA triggers apoptosis of p53 mutant murine mammary tumor cells through the mitochondrial pathway by targeting Bcl-2.     

Manganese superoxide dismutase inactivation during Fas (CD95)-mediated apoptosis in Jurkat T cells

Manganese superoxide dismutase (MnSOD, SOD2) is an essential primary antioxidant enzyme which converts superoxide radical to hydrogen peroxide within the mitochondrial matrix. MnSOD plays a prominent role in protection against many apoptotic stimuli. Its absence may therefore impair the cellular redox balance and enhance apoptosis. Our data show that in Jurkat T cells, following oligomerization of the Fas receptor, MnSOD is selectively degraded during apoptosis. In the presence of cycloheximide, an inhibitor of protein synthesis, the rates of cell death and MnSOD degradation were accelerated. Fas-induced MnSOD cleavage was partially inhibited in the presence of the pan-caspase inhibitor, z-VAD-fmk. MnSOD in the mitochondrial fractions was cleaved in vitro by treatment with the cytosolic fraction of Fas-activated cells. Moreover, two possible cleavage sites of recombinant hMnSOD by direct interaction with recombinant caspase-3 were noted. Cellular and mitochondrial factors were found to be necessary for the interaction. These factors include intracellular mobilization of calcium. Our data indicate that inactivation of MnSOD in receptor-mediated apoptosis by caspase-specific degradation would render the mitochondria sensitive to the steady-state production of superoxide, decrease the steady-state flux of H₂O₂, expedite the loss of mitochondrial function, and potentiate apoptosis.     

Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli

Sirtuins are NAD+-dependent protein deacetylase enzymes that are broadly conserved from bacteria to human, and have been implicated to play important roles in gene regulation, metabolism and longevity. cobB is a bacterial sirtuin that deacetylates acetyl-CoA synthetase (Acs) at an active site lysine to stimulate its enzymatic activity. Here, we report the structure of cobB bound to an acetyl-lysine containing non-cognate histone H4 substrate. A comparison with the previously reported archaeal and eukaryotic sirtuin structures reveals the greatest variability in a small zinc-binding domain implicated to play a particularly important role in substrate-specific binding by the sirtuin proteins. Comparison of the cobB/histone H4 complex with other sirtuin proteins in complex with acetyl-lysine containing substrates, further suggests that contacts to the acetyl-lysine side-chain and beta-sheet interactions with residues directly C-terminal to the acetyl-lysine represent conserved features of sirtuin-substrate recognition. Isothermal titration calorimetry studies were used to compare the affinity of cobB for a variety of cognate and non-cognate acetyl-lysine-bearing peptides revealing an exothermic reaction with relatively little discrimination between substrates. In contrast, similar studies employing intact acetylated Acs protein as a substrate reveal a binding reaction that is endothermic, suggesting that cobB recognition of substrate involves a burial of hydrophobic surface and/or structural rearrangement involving substrate regions distal to the acetyl-lysine-binding site. Together, these studies suggest that substrate-specific binding by sirtuin proteins involves contributions from the zinc-binding domain of the enzyme and substrate regions distal to the acetyl-lysine-binding site.     

Up-regulation of eEF1A2 promotes proliferation and inhibits apoptosis in prostate cancer

Background

eEF1A2 is a protein translation factor involved in protein synthesis, which possesses important function roles in cancer development. This study aims at investigating the expression pattern of eEF1A2 in prostate cancer and its potential role in prostate cancer development.

Methods

We examined the expression level of eEF1A2 in 30 pairs of prostate cancer tissues by using RT-PCR and immunohistochemical staining (IHC). Then we applied siRNA specifically targeting eEF1A2 to down-regulate its expression in DU-145 and PC-3 cells. Flow cytometer was used to explore apoptosis and Western-blot was used to detect the pathway proteins of apoptosis.

Results

Our results showed that the expression level of eEF1A2 in prostate cancer tissues was significantly higher compared to their corresponding normal tissues. Reduction of eEF1A2 expression in DU-145 and PC-3 cells led to a dramatic inhibition of proliferation accompanied with enhanced apoptosis rate. Western blot revealed that apoptosis pathway proteins (caspase3, BAD, BAX, PUMA) were significantly up-regulated after suppression of eEF1A2. More importantly, the levels of eEF1A2 and caspase3 were inversely correlated in prostate cancer tissues.

Conclusion

Our data suggests that eEF1A2 plays an important role in prostate cancer development, especially in inhibiting apoptosis. So eEF1A2 might serve as a potential therapeutic target in prostate cancer.     

Ruthenium methylimidazole complexes induced apoptosis in lung cancer A549 cells through intrinsic mitochondrial pathway

Ruthenium(II) methylimidazole complexes, with the general formula [Ru(MeIm)₄(N?N)]²⁺ (N?N = tip (RMC1), iip (RMC2), dppz (RMC3), dpq (RMC4); MeIm = 1-methylimidazole, tip = 2-(thiophene-2-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, iip = 2-(1H-imidazol-4-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, dppz = dipyrido[3,2-a:2′,3′-c]phenazine, dpq = pyrazino [2,3-f] [1,10]phenanthroline), were synthesized and characterized. As determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, these complexes displayed potent anti-proliferation activity against various cancer cells. RMC1 inhibited the growth of A549 (human lung adenocarcinoma) lung cells through induction of apoptotic cell death, as evidenced by the accumulation of cell population in sub-G1 phase. RMC1 also induced the depletion of mitochondrial membrane potential in A549 cells by regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Another experiment showed that Bid protein was also activated by RMC1, which implied that RMC1 could existed two pathways crosstalk, namely, have exogenous death receptor signaling pathway. These results demonstrated that RMC1 induced cancer cell death by acting on both mitochondrial and death receptor apoptotic pathways, suggesting that RMC1 could be a candidate for further evaluation as a chemotherapeutic agent against human cancers.     

Protein expression profiles in Saccharomyces cerevisiae during apoptosis induced by H2O2

We identified the proteins involved during apoptosis induced by H2O2 in Saccharomyces cerevisiae, and analyzed the global protein pattern by 2-DE. We analyzed classical parameters of apoptosis such as chromatin condensation, DNA fragmentation, and morphology changes of cells. Exposure of yeast cells to nonphysiological doses of peroxides decreases the expression (or increases degradation) of enzymes involved in protection against oxidative stress. This leads the yeast cells to a reduction of their antioxidant defense and makes the cells more prone to apoptosis. In our data the down expression of peroxiredoxin II and GST I, could induce a perturbation of mitochondrial function with an alteration of permeability of the membrane leading to the mitochondria-mediated apoptosis. Moreover, we identified a new spot of a classical glycolytic enzyme: the glyceraldehyde 3-phosphate dehydrogenase during apoptosis. It is known that GAPDH is an extremely abundant glycolytic enzyme with multiple functions and that its overexpression is evident during apoptosis induced by a variety of stimuli. Our results confirm that it is a major intracellular messenger mediating apoptotic death and that this new spot of GAPDH could be an intracellular sensor of oxidative stress during apoptosis induced by H2O2 in S. cerevisiae.     

Smac/DIABLO is required for effector caspase activation during apoptosis in human cells

Mitochondria play a pivotal role during stress-induced apoptosis as several proapoptotic proteins are released to the cytosol to activate caspases. Smac/DIABLO is one of the proapoptotic proteins released from the mitochondria and has been shown to inactivate IAPs. However, gene knockout studies in mice revealed a redundant role for Smac during development and cell death. By applying RNA interference-mediated loss of function approach, we demonstrate that Smac/DIABLO is required for the activation of effector but not initiator caspases during stress and receptor-mediated cell death in HeLa cells. Cells with reduced Smac resist apoptosis and retained clonogenicity. Our results suggest an obligatory role for Smac/DIABLO in these tumor cells during several pathways of apoptosis induction.     

Structure of Sir2Tm bound to a propionylated peptide

Lysine propionylation is a recently identified post‐translational modification that has been observed in proteins such as p53 and histones and is thought to play a role similar to acetylation in modulating protein activity. Members of the sirtuin family of deacetylases have been shown to have depropionylation activity, although the way in which the sirtuin catalytic site accommodates the bulkier propionyl group is not clear. We have determined the 1.8 Å structure of a Thermotoga maritima sirtuin, Sir2Tm, bound to a propionylated peptide derived from p53. A comparison with the structure of Sir2Tm bound to an acetylated peptide shows that hydrophobic residues in the active site shift to accommodate the bulkier propionyl group. Isothermal titration calorimetry data show that Sir2Tm binds propionylated substrates more tightly than acetylated substrates, but kinetic assays reveal that the catalytic rate of Sir2Tm deacylation of propionyl‐lysine is slightly reduced to acetyl‐lysine. These results serve to broaden our understanding of the newly identified propionyl‐lysine modification and the ability of sirtuins to depropionylate, as well as deacetylate, substrates.