Identification of a novel gene family,paralogs of inhibitor of apoptosis proteins present in plants,fungi, and animals

Only few orthologs of animal apoptosis regulators have been found in plants. Recently, the ectopic expression of mammalian inhibitor of apoptosis proteins (IAPs) has been shown to affect plant programmed cell death. Here, we identified two novel proteins homologous to Arabidopsis thaliana IAP-like protein (AtILP) 1 and 2 by applying an improved motif searching method. Furthermore, homologs of AtILP1 were found to occur as a novel gene family in other organisms such as fungi and animals including Homo sapiens (HsILP1). Like baculovirus IAP repeats (BIRs) in IAPs, ILPs contain two highly conserved BIR-like domains (BLDs) with a putative C2HC-type zinc finger. Phylogenetic analyses indicated that ILPs are putative paralogs of IAPs. Homology modeling revealed that the three-dimensional structure of BLD in HsILP1 is similar to that of BIR. Transient expression of HsILP1 resulted in inhibition of etoposide-induced apoptosis in HEK293 and HeLaS3 cells. These findings suggest that ILPs are conserved in a wide range of eukaryotes including plants, and that their functions are closely related to those of IAPs.     

Meiotic and epigenetic aberrations in Dnmt3L-deficient male germ cells

The DNA methyltransferase-like protein Dnmt3L is necessary for the establishment of genomic imprints in oogenesis and for normal spermatogenesis (Bourc'his et al., 2001; Hata et al., 2002). Also, a paternally imprinted gene, H19, loses DNA methylation in Dnmt3L-/- spermatogonia (Bourc'his and Bestor, 2004; Kaneda et al., 2004). To determine the reason for the impaired spermatogenesis in the Dnmt3L-/- testes, we have carried out a series of histological and molecular studies. We show here that Dnmt3L-/- germ cells were arrested and died around the early meiotic stage. A microarray-based gene expression-profiling analysis revealed that various gonad-specific and/or sex-chromosome-linked genes were downregulated in the Dnmt3L-/- testes. In contrast, expression of retrovirus-like intracisternal A-particle (IAP) sequences was upregulated; consistent with this observation, a specific IAP copy showed complete loss of DNA methylation. These findings indicate that Dnmt3L regulates germ cell-specific gene expression and IAP suppression, which are critical for male germ cell proliferation and meiosis.     

Development of Peptidomimetics Targeting IAPs

Inhibitor of apoptosis proteins (IAPs) such as XIAP subvert apoptosis by binding and inhibiting caspases. Because occupation of the XIAP BIR3 peptide binding pocket by Smac abolishes the XIAP–caspase 9 interaction, it is a proapoptotic event of great therapeutic interest. An assay for pocket binding was developed based on the displacement of Smac 7-mer from BIR3. Through the physical and biochemical analysis of a variety of peptides, we have determined the minimum sequence required for inhibition of the Smac–BIR3 interaction and detailed the dimensions and topology of the BIR3 peptide binding pocket. This work describes the structure–activity relationship (SAR) for peptide inhibitors of Smac-IAP binding.     

The RING Domain of TRAF2 Plays an Essential Role in the Inhibition of TNFα-Induced Cell Death but Not in the Activation of NF-κB

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK), as well as in inhibiting apoptosis induced by TNFα. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFα-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-ΔR) has been widely used as a dominant negative in transient overexpression systems to block TNFα-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-ΔR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFα-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells efficiently inhibited the TNFα-induced later phase of prolonged JNK activation, yet failed to inhibit TNFα-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFα and is essential for suppressing the noncanonical nuclear factor κB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFα-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.     

cIAP1/2 are involved in the radiosensitizing effect of birinapant on NSCLC cell line in vitro

Tumour radioresistance is a major problem for cancer radiation therapy. To identify the underlying mechanisms of this resistance, we used human non-small cell lung cancer (NSCLC) cell lines and focused on the Inhibitor of Apoptosis Protein (IAP) family, which contributes to tumourigenesis and chemoresistance. We investigated the possible correlation between radioresistance in six NSCLC cell lines and IAP protein levels and tested the radiosensitizing effect of birinapant in vitro, a molecule that mimics the second mitochondria-derived activator of caspase. We found that birinapant-induced apoptosis and inhibited the proliferation of NSCLC cells after exposure to radiation. These effects were induced by birinapant downregulation of cIAP protein levels and changes of cIAP gene expression. Overall, birinapant can inhibit tumour growth of NSCLC cell lines to ironizing radiation and act as a promising strategy to overcome radioresistance in NSCLC.     

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.     

Effects of changes in the shape of the intracellular action potential on the peak-to-peak ratio of single muscle fibre potentials

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet feasible, and consequently, knowledge about certain IAP characteristics of these IAPs is still limited. The ratio between the amplitudes of the second and first phases (the so-called peak-to-peak ratio, PPR) of a single fibre action potential (SFAP) is known to be closely related to the IAP profile. The PPR of experimentally recorded SFAPs has been found to be largely independent of changes in the fibre-to-electrode (radial) distance. The main goal of this paper is to analyze the effect of changes in different aspects of the IAP spike on the relationship between PPR and radial distance. Based on this analysis, we hypothesize about the characteristics of IAPs obtained experimentally. It was found that the sensitivity of the SFAP PPR to changes in radial distance is essentially governed by the duration of the IAP spike. Assuming that, for mammals, the duration of the IAP rising phase lies within the range 0.2-0.4 ms, we tentatively suggest that the duration of the IAP spike should be over approximately 0.75 ms, with the shape of the spike strongly asymmetric. These IAP characteristics broadly coincide with those observed in mammal IAPs.     

新的凋亡抑制因子Survivin的研究进展

Survivin是近年来发现的一种凋亡抑制因子,属于凋亡抑制蛋白（inhibitor of apoptosis proteins,IAP）家族的新成员;主要通过抑制caspase-3和caspase-7的活性而阻断细胞凋亡过程. Survivin选择性地表达于胚胎发育组织和大多数肿瘤组织,而正常成人终末分化组织中不表达. Survivin参与了细胞周期调控,与肿瘤的发生、发展和预后密切相关,可作为肿瘤治疗的新靶点.     

The anti-apoptotic ubiquitin conjugating enzyme BIRC6/BRUCE regulates autophagosome-lysosome fusion

The Inhibitor of Apoptosis (IAP) family member, Baculoviral IAP Repeat Containing 6 (BIRC6)/BRUCE is a ubiquitin conjugating E2 enzyme and a well-established anti-apoptosis regulator. However, its role in mammalian autophagy has not been shown. We identified BIRC6 as an important positive regulator of macroautophagy/autophagy by performing an siRNA screen targeting enzymes in the ubiquitin pathway. Compared to wild-type cells, BIRC6-deficient cells show accumulation of lipidated LC3B both at basal and starved conditions. Furthermore, BIRC6 deficiency blocks starvation-induced autophagic flux monitored by a tandem fluorescent autophagy sensor, mCherry-GFP-LC3B. Most strikingly, fusion of autophagosomes and lysosomes is blocked in BIRC6-deficient cells. BIRC6 colocalizes with the lysosomal protein LAMP2 in cells, and biochemically interacts with STX17 (syntaxin 17), which is a marker for completed autophagosomes. These data collectively suggest that BIRC6 bridges lysosomes and autophagosomes by interacting with these proteins. Because a deletion mutant of BIRC6 lacking the UBC domain partially rescues the autophagosome-lysosome fusion defect in BIRC6-deficient cells, a role of BIRC6 in this event is independent of its E2 catalytic activity.