Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes.
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Nitric oxide (NO) is a potent inhibitor of apoptosis in many cell types, including hepatocytes. We and others have described NO-dependent decreases in caspase activity in cells undergoing apoptosis. However, previous work has not determined whether NO disrupts the proteolytic processing and thus the activation of pro-caspases. Here we report that NO suppresses proteolytic processing and activation of multiple pro-caspases in intact cells, including caspase-3 and caspase-8. We found that both exogenous NO as well as endogenously produced NO via adenoviral inducible NO synthase gene transfer protected hepatocytes from tumor necrosid factor (TNF) alpha plus actinomycin D (TNFalpha/ActD)-induced apoptosis. Affinity labeling with biotin-VAD-fmk of all active caspase species in TNFalpha-mediated apoptosis identified four newly labeled spots (activated caspases) present exclusively in TNFalpha/ActD-treated cells. Both NO and the caspase inhibitor, Ac-DEVD-CHO, prevented the appearance of the four newly labeled spots or active caspases. Immunoanalysis of affinity labeled caspases demonstrated that caspase-3 was the major effector caspase. Western blot analysis also identified the activation of caspase-8 in the TNFalpha/ActD-treated cells, and the activation was suppressed by NO. Furthermore, NO inhibited several other events associated with caspase activation in cells, including release of cytochrome c from mitochondria, decrease in mitochondrial transmembrane potential, and cleavage of poly(ADP-ribose) polymerase in TNFalpha/ActD-treated cells. These findings indicate the involvement of multiple caspases in TNFalpha-mediated apoptosis in hepatocytes and establish the capacity of NO to inhibit not only active caspases but also caspase activation. (+info)
Evolution of antigen-specific T cell receptors in vivo: preimmune and antigen-driven selection of preferred complementarity-determining region 3 (CDR3) motifs.
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Antigen (Ag)-driven selection of helper T cells (Th) in normal animals has been difficult to study and remains poorly understood. Using the major histocompatibility complex class II- restricted murine response to pigeon cytochrome c (PCC), we provide evidence for both preimmune and Ag-driven selection in the evolution of Ag-specific immunity in vivo. Before antigenic challenge, most Valpha11(+)Vbeta3(+) Th (70%) express a critical complementarity-determining region 3 (CDR3) residue (glutamic acid at TCR-alpha93) associated with PCC peptide contact. Over the first 5 d of the primary response, PCC-responsive Valpha11(+)Vbeta3(+) Th expressing eight preferred CDR3 features are rapidly selected in vivo. Clonal dominance is further propagated through selective expansion of the PCC-specific cells with T cell receptor (TCR) of the "best fit." Ag-driven selection is complete before significant emergence of the germinal center reaction. These data argue that thymic selection shapes TCR-alpha V region bias in the preimmune repertoire; however, Ag itself and the nongerminal center microenvironment drive the selective expansion of clones with preferred TCR that dominate the response to Ag in vivo. (+info)
Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis.
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BACKGROUND: The importance of free radical homeostasis and apoptosis in normal and diseased hearts and their interrelationships are poorly defined. We tested whether reactive oxygen species can trigger apoptosis in cardiomyocytes, and we explored the underlying pathways. METHODS AND RESULTS: A cell culture model of isolated cardiac cells and different reactive oxygen species (ROS)-generating systems were used. Apoptosis became evident when cardiomyocytes were exposed to either H2O2 or superoxide anion (O2-). Both H2O2- and O2--induced apoptosis of cardiomyocytes were associated with an increase in p53 protein content, whereas protein levels of Bax and Bcl-2 were unaltered. H2O2, but not O2-, induced an increase in the protein content of Bad. Furthermore, H2O2 elicited translocation of Bax and Bad from cytosol to mitochondria, where these factors formed heterodimers with Bcl-2, which was followed by the release of cytochrome c, activation of CPP32, and cleavage of poly(ADP-ribose) polymerase. Interestingly, this pathway was not activated by O2-. Instead, O2- used Mch2alpha to promote the apoptotic pathway, as revealed by the activation of Mch2alpha and the cleavage of its substrate, lamin A. CONCLUSIONS: Taken together, these results indicate that ROS may play an important pathophysiological role in cardiac diseases characterized by apoptotic cell death and suggest that different ROS-induced activations of the apoptotic cell death program in cardiomyocytes involve distinct signaling pathways. (+info)
Catalysis of peptide dissociation from class II MHC-peptide complexes.
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Certain peptides such as dynorphin A [dynA-(1-13)] enhance the release of antigenic peptides bound to class II MHC molecules at neutral pH. This enhanced release has been termed push off. Previous work has shown that the antigenic pigeon cytochrome c peptide PCC-(89-104) has at least two conformational isomers when bound to the class II MHC protein I-Ek. We have accordingly studied the push off of PCC-(89-104) from the complex PCC-(89-104)/I-Ek to see whether these isomeric conformations are distinguished by the push-off effect. A comparison of the association and dissociation kinetics of PCC-(89-104)/I-Ek in the presence of dynA-(1-13) shows that dynA-(1-13) does not simply replace PCC-(89-104) but rather acts catalytically. The major product is peptide-free I-Ek, which is receptive to further peptide binding. Evidence is presented that a two peptide-one MHC complex is formed in solution. This ternary complex represents the first step of the mechanism of push off. 19F NMR data are presented that indicate that dynA-(1-13) interacts specifically with only one of the two isomeric complexes of PCC-(89-104) and I-Ek. A push-off mechanism is proposed in which dynA-(1-13) binds outside the peptide binding groove. In a second step, the dissociation of one of the two isomers is specifically enhanced. Thus the push-off effect may be useful for identifying conformational isomers and for separating them. (+info)
Purified photoproducts of merocyanine 540 trigger cytochrome C release and caspase 8-dependent apoptosis in human leukemia and melanoma cells.
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If the interplay between caspase proteases and mitochondria decide the fate of the cell during apoptosis, they may constitute useful molecular targets for novel drug design. We have shown that photoactivated merocyanine 540 (pMC540) triggers caspase-mediated apoptosis in HL60 leukemia and M14 melanoma cells. Because pMC540 is a mixture of photoproducts, we set out to purify the biologically active component(s) from this mixture and to investigate their ability to directly activate intracellular caspases and/or trigger mitochondrial events associated with apoptosis. Two photoproducts, namely C1 and C2, purified and characterized by mass spectroscopy and nuclear magnetic resonance (NMR) analysis, effectively induced apoptosis in HL60 and M14 cells. Interestingly, both C1 and C2 induced non-receptor-dependent activation of caspase 8, which was responsible for the downstream activation of caspase 3 and cell death. Both compounds induced the release of cytochrome C from mitochondria of tumor cells and from purified rat liver mitochondria; however, different mechanisms were operative in cytochrome C translocation in response to C1 or C2. C1-induced cytochrome C release was mediated by the mitochondrial permeability transition (MPT) pore and accompanied by a decrease in mitochondrial transmembrane potential (triangle uppsim), whereas cytochrome C release in response to C2 was independent of MPT pore opening. These findings do not exclude the possibility that changes in mitochondrial triangle uppsim are critical for apoptosis in some instances, but support the notion that this may not be a universal step in the apoptotic process. Thus, identification of two novel anticancer agents that directly activate effector components of the apoptotic pathway could have potential implications for the development of newer chemotherapeutic drugs. (+info)
c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release.
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Expression of c-Myc sensitizes cells to a wide range of pro-apoptotic stimuli. We here show that this pro-apoptotic effect is mediated through release of mitochondrial holocytochrome c into the cytosol. First, activation of c-Myc triggers release of cytochrome c from mitochondria. This release is caspase-independent and blocked by the survival factor IGF-1. Second, c-Myc-induced apoptosis is blocked by microinjection of anticytochrome c antibody. In addition, we show that microinjection of holocytochrome c mimics the effect of c-Myc activation, sensitizing cells to DNA damage and to the CD95 pathway. Both p53 and CD95/Fas signaling have been implicated in c-Myc-induced apoptosis but neither was required for c-Myc-induced cytochrome c release. Nonetheless, inhibition of CD95 signaling in fibroblasts did prevent c-Myc-induced apoptosis, apparently by obstructing the ability of cytosolic cytochrome c to activate caspases. We conclude that c-Myc promotes apoptosis by causing the release of cytochrome c, but the ability of cytochrome c to activate apoptosis is critically dependent upon other signals. (+info)
Caspases induce cytochrome c release from mitochondria by activating cytosolic factors.
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We investigated the ability of caspases (cysteine proteases with aspartic acid specificity) to induce cytochrome c release from mitochondria. When Jurkat cells were induced to undergo apoptosis by Fas receptor ligation, cytochrome c was released from mitochondria, an event that was prevented by the caspase inhibitor, zVAD-fmk (zVal-Ala-Asp-CH2F). Purified caspase-8 triggered rapid cytochrome c release from isolated mitochondria in vitro. The effect was indirect, as the presence of cytosol was required, suggesting that caspase-8 cleaves and activates a cytosolic substrate, which in turn is able to induce cytochrome c release from mitochondria. The cytochrome c releasing activity was not blocked by caspase inhibition, but was antagonized by Bcl-2 or Bcl-xL. Caspase-8 and caspase-3 cleaved Bid, a proapoptotic Bcl-2 family member, which gains cytochrome c releasing activity in response to caspase cleavage. However, caspase-6 and caspase-7 did not cleave Bid, although they initiated cytochrome c release from mitochondria in the presence of cytosol. Thus, effector caspases may cleave and activate another cytosolic substrate (other than Bid), which then promotes cytochrome c release from mitochondria. Mitochondria significantly amplified the caspase-8 initiated DEVD-specific cleavage activity. Our data suggest that cytochrome c release, initiated by the action of caspases on a cytosolic substrates, may act to amplify a caspase cascade during apoptosis. (+info)
Ligand exchange during unfolding of cytochrome c.
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The productive folding pathway of cytochrome c passes through an obligatory HW intermediate in which the heme is coordinated by a solvent water molecule and a native ligand, His-18, prior to the formation of the folded HM state with both the native His-18 and Met-80 heme coordination. Two off pathway intermediates, a five-coordinated state (5C) and a bis-histidine state (HH), were also identified during the folding reaction. In the present work, the thermodynamics and the kinetics of the unfolding reaction of cytochrome c were investigated with resonance Raman scattering, tryptophan fluorescence spectroscopy, and circular dichroism. The objective of these experiments was to determine if the protein opens up and diverges into the differing heme ligation states through a many pathway mechanism or if it passes through intermediate states analogous to those observed during the folding reaction. Equilibrium unfolding results indicate that, in contrast to 5C, the stability of HH with respect to HW decreases as the concentration of GdnHCl increases. The difference in their response to the denaturant indicates that the polypeptide structure of 5C is relatively loose as compared with HH in which the polypeptide is misfolded. Time-resolved resonance Raman measurements show that strikingly similar ligand exchange reactions occur during unfolding as were observed during folding. Combined with fluorescence data, a kinetic model is proposed in which local structural rearrangements controlled by heme ligand exchange reactions appear prior to the global relaxation of the polypeptide chain. (+info)