Reduced Apaf-1 levels in cardiomyocytes engage strict regulation of apoptosis by endogenous XIAP

Overexpression studies have identified X-linked inhibitor of apoptosis protein (XIAP) as a potent inhibitor of caspases. However, the exact function of endogenous XIAP in regulating mammalian apoptosis is less clear. Endogenous XIAP strictly regulates cytochrome c-dependent caspase activation in sympathetic neurons but not in many mitotic cells. We report that postmitotic cardiomyocytes, unlike fibroblasts, are remarkably resistant to cytosolic microinjection of cytochrome c. The cardiomyocyte resistance to cytochrome c is mediated by endogenous XIAP, as XIAP-deficient cardiomyocytes die rapidly with cytosolic cytochrome c alone. Importantly, we found that cardiomyocytes, like neurons, have markedly reduced Apaf-1 levels and that this decrease in Apaf-1 is directly linked to the tight regulation of caspase activation by XIAP. These data identify an important function of XIAP in cardiomyocytes and point to a striking similarity in the regulation of apoptosis in postmitotic cells.     

Cell division regulation by BIR1, a member of the inhibitor of apoptosis family in yeast

The inhibitor of apoptosis (IAP) gene family comprises molecules that block the activity of pro-apoptotic caspase proteases. Paradoxically, yeasts contain IAP proteins but no caspases and no apoptotic program. To determine the function of these proteins in vivo, we disrupted the BIR1 gene, encoding the only known IAP in yeast Saccharomyces cerevisiae. Sporulation of heterozygous diploids yielded no viable mutant haploids, indicating that BIR1 is an essential gene. By flow cytometry, some heterozygous mutants were polyploid accumulating >4 N DNA content. These cells exhibited a 20-40% reduction in growth rate, which was rescued by plasmid-borne over-expression of BIR1 but not by its human counterpart, survivin. Deletion analysis revealed that the N-terminal domain of Bir1, containing the conserved baculovirus IAP repeat, was able to partially complement the cell growth defect caused by BIR1 deletion. Moreover, the full-length and truncated forms of Bir1 accelerated cell division in wild-type cells. Finally, BIR1 heterozygous mutants exhibited grossly altered cell morphology with misshapen or abnormally long buds connected to an unusually large mother cell. These findings identify a novel function of IAP proteins in the pleiotropic control of cell division, in addition to their role in the suppression of apoptosis.     

Conjugation of a novel Apaf-1 inhibitor to peptide-based cell-membrane transporters: effective methods to improve inhibition of mitochondria-mediated apoptosis

We have identified a family of peptoids that inhibits in vitro the activity of the apoptosome, a macromolecular complex that activates mitochondrial-dependent apoptosis pathways. The analysis of peptide-based cell compatible delivery systems of the most active peptoid is presented. The active peptoid was then fused to cell penetrating peptides (CPP) as penetratin (PEN-peptoid) and HIV-1 TAT (TAT-peptoid). PEN-peptoid showed greater cell viability and as a consequence better efficiency as an apoptosis inhibitor than the TAT-peptoid. The intracellular trafficking of both inhibitors was studied by flow cytometry and confocal fluorescence microscopy. Finally, the influence of the cargo (peptoid) molecules on the conformational behavior of the CPP in buffers and in membrane mimetic environments was analyzed using circular dichroism (CD) spectroscopy.     

Negative regulation of the Apaf-1 apoptosome by Hsp70

Release of cytochrome c from mitochondria by apoptotic signals induces ATP/dATP-dependent formation of the oligomeric Apaf-1-caspase-9 apoptosome. Here we show that the documented anti-apoptotic effect of the principal heat-shock protein, Hsp70, is mediated through its direct association with the caspase-recruitment domain (CARD) of Apaf-1 and through inhibition of apoptosome formation. The interaction between Hsp70 and Apaf-1 prevents oligomerization of Apaf-1 and association of Apaf-1 with procaspase-9. On the basis of these results, we propose that resistance to apoptosis exhibited by stressed cells and some tumours, which constitutively express high levels of Hsp70, may be due in part to modulation of Apaf-1 function by Hsp70.     

Heat shock proteins: essential proteins for apoptosis regulation

Many different external and intrinsic apoptotic stimuli induce the accumulation in the cells of a set of proteins known as stress or heat shock proteins (HSPs). HSPs are conserved proteins present in both prokaryotes and eukaryotes. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and by preventing their aggregation. HSPs have a protective function, that is they allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several of these proteins have demonstrated to directly interact with components of the cell signalling pathways, for example those of the tightly regulated caspase-dependent programmed cell death machinery, upstream, downstream and at the mitochondrial level. HSPs can also affect caspase-independent apoptosis-like process by interacting with apoptogenic factors such as apoptosis-inducing factor (AIF) or by acting at the lysosome level. This review will describe the different key apoptotic proteins interacting with HSPs and the consequences of these interactions in cell survival, proliferation and apoptotic processes. Our purpose will be illustrated by emerging strategies in targeting these protective proteins to treat haematological malignancies.     

Tankyrase 1 interacts with Mcl-1 proteins and inhibits their regulation of apoptosis

Mcl-1L (myeloid cell leukemia-1 long) is an antiapoptotic Bcl-2 family protein discovered as an early induction gene during leukemia cell differentiation. Previously, we identified Mcl-1S (short) as a short splicing variant of the Mcl-1 gene with proapoptotic activity. To identify Mcl-1-interacting proteins, we performed yeast two-hybrid screening and found cDNAs encoding tankyrase 1. This protein possesses poly(ADP-ribose) polymerase activity and presumably facilitates the turnover of substrates following ADP-ribosylation. In yeast and mammalian cells, tankyrase 1 interacts with both Mcl-1L and Mcl-1S, but does not bind to other Bcl-2 family proteins tested. Analysis of truncated tankyrase 1 mutants indicated that the first 10 ankyrin repeats are involved in interaction with Mcl-1. In the N terminus of Mcl-1, a stretch of 25 amino acids is sufficient for binding to tankyrase 1. Overexpression of tankyrase 1 antagonizes both Mcl-1L-mediated cell survival and Mcl-1S-induced cell death. Furthermore, coexpression of tankyrase 1 with Mcl-1L or Mcl-1S decreased the levels of Mcl-1 proteins. Although tankyrase 1 down-regulates Mcl-1 protein expression, no ADP-ribosylation of Mcl-1 was detected. In contrast, overexpression of Mcl-1 proteins suppressed the ADP-ribosylation of the telomeric repeat binding factor 1, another tankyrase 1-interacting protein. Thus, interaction of Mcl-1L and Mcl-1S with tankyrase 1 could serve as a unique mechanism to decrease the expression of these Bcl-2 family proteins, thereby leading to the modulation of the apoptosis pathway.     

Differential regulation of the apoptotic machinery during megakaryocyte differentiation and platelet production by inhibitor of apoptosis protein Livin

Livin is a member of the inhibitor of apoptosis proteins (IAP) family of intracellular antiapoptotic proteins that act by binding and inhibiting caspases. Upon strong apoptotic stimuli, it is then specifically cleaved by caspases to produce a truncated protein (tLivin) with a paradoxical proapoptotic activity. Intriguingly, we have detected robust protein levels of Livin in normal mature bone marrow megakaryocyte (MK) and platelets. To evaluate the potential role of Livin in thrombopoiesis, we used the human BCR-ABL+ cell line, LAMA-84, and cord blood CD34+ cells to induce differentiation toward MKs. Upon differentiation, induced by phorbol myristate acetate and concurrent with increase in Livin protein expression, LAMA-84 cells formed functional platelet-like particles. Livin overexpression in CD34+ progenitor cells induced higher endoreplication in the MKs generated. Furthermore, overexpression of Livin increased the ability of both primary MKs and differentiated LAMA-84 cells to produce functional platelets. In the differentiated LAMA-84 cells, we observed accumulation of proapoptotic tLivin concomitant with increased caspase-3 activity. Downregulation of Livin with small interfering RNA in both leukemic and primary MK cells decreased their ability to produce functional platelets. We suggest that Livin has a role in thrombopoiesis by regulating the apoptotic and antiapoptotic balance in MK endoreplication and platelet production.     

热激蛋白对细胞凋亡的调节作用

细胞凋亡是生物发育过程中或在正常生理状态下清除衰老及受损细胞的一种普遍现象。细胞凋亡的发生受胞外或胞内的多种刺激源所诱导,其中热激蛋白是细胞凋亡的调控因子之一。本文着重讨论了热激蛋白在细胞凋亡调节中所发挥的作用。     

Cell death: drosophila Apaf-1 - no longer in the (d)Ark

The recent discovery and characterization of Ark, the Drosophila homolog of the mammalian cell-death adaptor protein Apaf-1, have revealed that, like Apaf-1, this protein is important in multiple apoptosis pathways. The new findings also suggest that cell death in flies is very similar to that in mammals after all.     

Insufficient Apaf-1 expression in early stages of neural differentiation of human embryonic stem cells might protect them from apoptosis

Recent evidence suggests that mitochondrial apoptosis regulators and executioners may regulate differentiation, without being involved in cell death. However, the involved factors and their roles in differentiation and apoptosis are still not fully determined. In the present study, we compared mitochondrial pathway of cell death during early neural differentiation from human embryonic stem cells (hESCs). Our results demonstrated that ROS generation, cytosolic cytochrome c release, caspases activation and rise in p53 protein level occurred upon either neural or apoptosis induction in hESCs. However, unlike apoptosis, no remarkable increase in apoptotic protease activating factor-1 (Apaf-1) level at early stages of differentiation was observed. Also the caspase-like activity of caspase-9 and caspase-3/7 were seen less than apoptosis. The results suggest that low levels of Apaf-1 as an adaptor protein might be considered as a possible regulatory barrier by which differentiating cells control cell death upon rise in ROS production and cytochrome c release from mitochondria. Better understanding of mechanisms via which mitochondria-mediated apoptotic pathway promote neural differentiation can result in development of novel therapeutic approaches.     

Nucling recruits Apaf-1/pro-caspase-9 complex for the induction of stress-induced apoptosis

Nucling is a novel protein isolated from murine embryonal carcinoma cells with an up-regulated expression during cardiac muscle differentiation. We show here that Nucling was up-regulated by proapoptotic stimuli and important for the induction of apoptosis after cytotoxic stress. We further demonstrated that overexpressed Nucling was able to induce apoptosis. In Nucling-deficient cells, the expression levels of Apaf-1 and cytochrome c, which are the major components of an apoptosis-promoting complex named apoptosome, were both down-regulated under cellular stress. A deficiency of Nucling also conferred resistance to apoptotic stress on the cell. After UV irradiation, Nucling was shown to reside in an Apaf-1/pro-caspase-9 complex, suggesting that Nucling might be a key molecule for the formation and maintenance of this complex. Nucling induced translocation of Apaf-1 to the nucleus, thereby distributing the Nucling/Apaf-1/pro-caspase-9 complex to the nuclear fraction. These findings suggest that Nucling recruits and transports the apoptosome complex during stress-induced apoptosis.