Mcl-1 fragment induces apoptosis through direct interaction with Bax

Mcl-1 full-length (Mcl-1^1-350), a tightly regulated protein, plays an important role in protecting cells against apoptosis. Cleavage of Mcl-1 at Asp127 by caspase (Mcl-1^C1) contributes to the regulation of Mcl-1 expression, but its pro-apoptotic function remains controversial. Here, we reported that Mcl-1^128-350 expression induced caspase-dependent apoptosis. We demonstrated that Mcl-1^128-350 but not Mcl-1^1-350 interacts with Bax. This interaction required an intact BH3 Mcl-1^128-350 domain and leads to Bax activation and translocation to mitochondria. The silencing of Bax, but not of Bak, prevented Mcl-1^128-350 induced apoptosis. In conclusion, Mcl-1^128-350 exerts a pro-apoptotic function governed by its capacity to interact with Bax.

Structured summary

MINT-7306752: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0915) with BAK (uniprotkb:Q16611) by anti tag coimmunoprecipitation (MI:0007)MINT-7306728: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0914) with BAX (uniprotkb:Q07812) and BAK (uniprotkb:Q16611) by anti tag coimmunoprecipitation (MI:0007)MINT-7307171: F1 ATPase (uniprotkb:Q5TC12), Mcl-1 (uniprotkb:Q07820) and BAX (uniprotkb:Q07812) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)     

Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1(+)/CD127(-) HCV-specific CD8(+) cells targeting the virus in chronic hepatitis C virus infection

PD-1 molecule promotes anergy and IL-7 receptor (CD127) induces an anti-apoptotic effect on T cells. Correlation between PD-1/CD127 phenotype and hepatitis C virus (HCV)-specific CD8⁺ cell reactivity in resolved infection (RI) after treatment and persistent HCV-infection (PI) was analysed. Directly ex vivo, PD-1 and CD127 expression on HCV-specific CD8⁺ cells displayed a positive and negative correlation, respectively with viraemia. Proliferation after stimulation on PD-1⁻/CD127⁺ cells from RI cases was preserved, while it was impaired on PD-1⁺/CD127⁻ cells from PI patients. PD1⁺/CD127⁺ population was observed in PI, and these maintained expansion ability but they did not target the virus. Frequency of PI cases with HCV-specific CD8⁺ cell proliferation increased after anti-PD-L1 and anti-apoptotic treatment. Bim expression on HCV-specific CD8⁺ cells from PI patients was enhanced. In conclusion, during chronic HCV infection non-reactive HCV-specific CD8⁺ cells targeting the virus are PD-1⁺/CD127⁻/Bim⁺ and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in vitro reactivity.     

YCA1 participates in the acetic acid induced yeast programmed cell death also in a manner unrelated to its caspase-like activity

Yeast cells lacking the metacaspase-encoding gene YCA1 (Δyca1) were compared with wild-type (WT) cells with respect to the occurrence, nature and time course of acetic-acid triggered death. We show that Δyca1 cells undergo programmed cell death (PCD) with a rate lower than that of the WT and that PCD in WT cells is caused at least in part by the caspase activity of Yca1p. Since in Δyca1 cells this effect is lost, but z-VAD-fmk does not prevent both WT and Δyca1 cell death, PCD in WT cells occurs via a Yca1p caspase and a non-caspase route with similar characteristics.     

Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1

To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Δyca1), cytochrome c (Δcyc1,7) and both (Δcyc1,7Δyca1) were compared for AA-PCD occurrence, hydrogen peroxide (H₂O₂) production and caspase activity. AA-PCD occurs in Δcyc1,7 and Δcyc1,7Δyca1 cells slower than in wt, but similar to that in Δyca1 cells, in which no cytochrome c release occurs. Both H₂O₂ production and caspase activation occur in these cells with early and extra-activation in Δcyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.     

Extracellular ADP prevents neuronal apoptosis via activation of cell antioxidant enzymes and protection of mitochondrial ANT-1

Apoptosis in neuronal tissue is an efficient mechanism which contributes to both normal cell development and pathological cell death. The present study explores the effects of extracellular ADP on low [K⁺]-induced apoptosis in rat cerebellar granule cells. ADP, released into the extracellular space in brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine. The findings reported in this paper demonstrate that ADP inhibits the proapoptotic stimulus supposedly via: i) inhibition of ROS production during early stages of apoptosis, an effect mediated by its interaction with cell receptor/s. This conclusion is validated by the increase in SOD and catalase activities as well as by the GSSG/GSH ratio value decrease, in conjunction with the drop of ROS level and the prevention of the ADP protective effect by pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a novel functionally selective antagonist of purine receptor; ii) safeguard of the functionality of the mitochondrial adenine nucleotide-1 translocator (ANT-1), which is early impaired during apoptosis. This effect is mediated by its plausible internalization into cell occurring as such or after its hydrolysis, by means of plasma membrane nucleotide metabolizing enzymes, and resynthesis into the cell. Moreover, the findings that ADP also protects ANT-1 from the toxic action of the two Alzheimer's disease peptides, i.e. Aβ1–42 and NH₂htau, which are known to be produced in apoptotic cerebellar neurons, further corroborate the molecular mechanism of neuroprotection by ADP, herein proposed.     

Wnt11/Fgfr1b cross-talk modulates the fate of cells in palate development

Various cellular and molecular events underlie the elevation and fusion of the developing palate that occurs during embryonic development. This includes convergent extension, where the medial edge epithelium is intercalated into the midline epithelial seam. We examined the expression patterns of Wnt11 and Fgfr1b - which are believed to be key factors in convergent extension - in mouse palate development. Wnt-11 overexpression and beads soaked in SU5402 (an Fgfr1 inhibitor) were employed in in vitro organ cultures. The results suggested that interactions between Wnt11 and Fgfr1b are important in modulating cellular events such as cell proliferation for growth and apoptosis for fusion. Moreover, the Wnt11 siRNA results showed that Wnt11-induced apoptosis was necessary for palatal fusion. In summary, Fgfr1b induces cell proliferation in the developing palate mesenchyme so that the palate grows and contacts each palatal shelf, with negative feedback of Fgfs triggered by excessive cell proliferation then inhibiting the expression of Fgfr1b and activating the expression of Wnt11 to fuse each palate by activating apoptosis.     

Trichomonas vaginalis: reactive oxygen species mediates caspase-3 dependent apoptosis of human neutrophils

There are many neutrophils in the vaginal discharge from women infected with Trichomonas vaginalis. The aim of our study was to determine whether human neutrophil apoptosis may be regulated by reactive oxygen species (ROS) in response to trichomonads infection. Incubation of human neutrophils with live trichomonads caused marked receptor shedding of CD16, decrease of mitochondrial membrane potential (MMP) and caspase-3 activation in human neutrophils. These proapoptotic effects of T. vaginalis on neutrophils were inhibited by pretreatment of neutrophils with an inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI), suggesting an important role of intracellular ROS accumulation in T. vaginalis-triggered apoptosis. Indeed, large amounts of ROS levels were detected in neutrophils incubated with live trichomonads, and were also effectively inhibited by DPI. However, pan-caspase inhibitor z-VAD-fmk or caspase-3 inhibitor z-DEVD-fmk did not affect T. vaginalis-induced ROS generation in neutrophils. These results suggest that ROS-dependent caspase-3 activation plays an important role in apoptosis of human neutrophils induced by T. vaginalis.     

PKC activation contributes to caspase-mediated eIF2α phosphorylation and cell death

Back ground

Stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2α), involved in translation, promotes cell suicide or survival. Since multiple signaling pathways are implicated in cell death, the present study has analyzed the importance of PKC activation in the stress-induced eIF2α phosphorylation, caspase activation and cell death in the ovarian cells of Spodoptera frugiperda (Sf9) and in their extracts.

Methods

Cell death is analyzed by flow cytometry. Caspase activation is measured by Ac-DEVD-AFC hydrolysis and also by the cleavage of purified recombinant PERK, an endoplasmic reticulum-resident eIF2α kinase. Status of eIF2α phosphorylation and cytochrome c levels are analyzed by western blots.

Results

PMA, an activator of PKC, does not promote cell death or affect eIF2α phosphorylation. However, PMA enhances late stages of UV-irradiation or cycloheximide-induced caspase activation, eIF2α phosphorylation and apoptosis in Sf9 cells. PMA also enhances cytochrome c-induced caspase activation and eIF2α phosphorylation in cell extracts. These changes are mitigated more efficiently by caspase inhibitor, z-VAD-fmk, than by calphostin, an inhibitor of PKC. In contrast, tunicamycin-induced eIF2α phosphorylation that does not lead to caspase activation or cell death is unaffected by PMA, z-VAD-fmk or by calphostin.

Conclusions

While caspase activation is a cause and consequence of eIF2α phosphorylation, PKC activation that follows caspase activation further enhances caspase activation, eIF2α phosphorylation, and cell death in Sf9 cells.

General significance

Caspases can activate multiple signaling pathways to enhance cell death.     

Involvement of Ceramide in the Mechanism of Cr(VI)-induced Apoptosis of CHO Cells

Mitochondria reduce Cr(VI) to Cr(V) with concomitant generation of reactive oxygen species, thereby exhibiting cytotoxic effects leading to apoptosis in various types of cells. To clarify the mechanism by which Cr(VI) induces apoptosis, we examined the effect of Cr(VI) on Chinese hamster ovary (CHO) cells. Cr(VI) increased cellular levels of ceramide by activating acid sphingomyelinase (ASMase) and inhibiting the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt). Cr(VI) also induced cyclosporin A- and trifluoperazine-sensitive depolarization of mitochondria and activated caspase-3, 8 and 9, thereby causing fragmentation of cellular DNA. The presence of desipramine, an inhibitor of ASMase, and membrane permeable pCPT-cAMP suppressed the Cr(VI)-induced activation of caspases and DNA fragmentation. These results suggested that accumulation of ceramide play an important role in the Cr(VI)-induced apoptosis of CHO cells through activation of mitochondrial membrane permeability transition.     

ROCK-II-induced membrane blebbing and chromatin condensation require actin cytoskeleton

Ectopic expression of ROCK II (Rho kinase II or ROKalpha), an effector of Rho GTPase, induces membrane blebbing and chromatin condensation. ROCK II can induce membrane blebbing in the presence of the caspase inhibitor z-VAD-fmk or in caspase-3-deficient MCF-7 cells, indicating that the activation of caspases is not required. ROCK-II-induced membrane blebbing, however, is reversed by the myosin light chain kinase inhibitor ML-7 or cytochalasin D. In addition, the expression of a constitutively activated form of cofilin (S3A-cofilin) suppresses both membrane blebbing and chromatin condensation in ROCK II expressing cells. These findings suggest that the activation of actin-myosin contractility is responsible for membrane blebbing and chromatin condensation and implicate ROCK II as a potential mediator of the morphological changes associated with apoptosis.