To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.

In vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis. Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase.

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insectGrim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 13 converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAFI and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

We have generated rat monoclonal antibodies that specifically recognise caspase-2 from many species, including mouse, rat and humans. Using these antibodies, we have investigated caspase-2 expression, subcellular localisation and processing. We demonstrate that caspase-2 is expressed in most tissues and cell types. Cell fractionation and immunohistochemistry experiments show that caspase-2 is found in the nuclear and cytosolic fractions, including a significant portion present in the Golgi complex. We found that caspase-2 is processed in response to many apoptotic stimuli but experiments with caspase-2 deficient mice demonstrated that it is not required for apoptosis of thymocytes or dorsal root ganglia (DRG) neurons in response to a variety of cytotoxic stimuli. Caspase-2 processing does not occur in thymocytes lacking Apaf-1 or caspase-9, suggesting that in this cell type, activation of caspase-2 occurs downstream of apoptosome formation.

Inhibitor of apoptosis (IAP) proteins, which bind to caspases via their baculoviral IAP repeat domains, also bear RING domains that enable them to promote ubiquitylation of themselves and other interacting proteins. Here we show that the RING domain of cIAP1 allows it to bind directly to the RING of X-linked IAP, causing its ubiquitylation and degradation by the proteasome, thus revealing a mechanism by which IAPs can regulate their abundance. Expression of a construct containing the RING of cellular IAP1 was able to deplete melanoma cells of endogenous X-linked IAP, promoted apoptosis, and also markedly reduced their clonogenicity when treated with cisplatin. Cross control of protein levels by RING domains may therefore enable their levels to be manipulated therapeutically.apoptosis ͉ ubiquitin ͉ homeostasis ͉ E3 ligase I nhibitor of apoptosis (IAP) proteins were initially identified in baculoviruses, where they prevent defensive apoptosis of the host cell (1), thereby increasing the time available for viral replication. Cellular IAP (cIAP) homologues, which all bear one to three baculoviral IAP repeat (BIR) domains, have been identified in yeasts and metazoans. Those that bear a RING domain in addition to BIR domains [X-linked IAP (XIAP), cIAP1, cIAP2, and ML-IAP͞Livin] appear to function as cell death inhibitors (reviewed in ref.2).The RING domains of IAPs can act as E3 ubiquitin ligases to promote the ubiquitylation of associated proteins such as TNF receptor-associated factors (TRAFs), Smac͞Diablo, and caspases (3-6). However, the importance of the RING domain for the antiapoptotic activity of the IAPs is unclear; on the one hand, a RING-less DIAP1 protein overexpressed in Drosophila had increased antiapoptotic activity (7); on the other hand, alleles of DIAP1 with mutations in the RING finger are null for Reaperinduced cell death, although more potent at blocking Hid-induced cell death (5,8,9).Our initial experiments showed that cIAP1 and XIAP can heterodimerize via a RING-RING interaction, but we also observed that expression of a stably integrated cIAP1 gene caused a specific reduction in the abundance of endogenous XIAP. Deletion studies revealed that the RING finger of cIAP1 was necessary and sufficient to cause loss of XIAP in a proteasome-dependent manner. cIAP1 RING-stimulated depletion of XIAP was seen in several cell types and associated with greatly increased sensitivity of several melanoma cells to cisplatin. Because several other E3 ligases such as BRCA1, BARD1, and RAG1 can interact via their RING fingers, it is possible that other RING-containing E3 ligases act to regulate the abundance of each other following heterodimerization. In this regard, it is striking that the E3 ligase activity of BRCA1 is greatly enhanced by heterodimerization with BARD1 (10, 11), suggesting a possible mechanism for homeostatic control of protein levels by RING domains. Materials and MethodsTransfections and Constructs. The complete sequence of all constructs used can be obtained upon request. Full length IAPs, RIN...

IntroductionPolycomb group (PcG) proteins are epigenetic repressors important in the maintenance of transcriptional silencing. PcG proteins exist in 3 distinct complexes: polycomb repressive complexes (PRC)1, PRC2 and pleiohomeotic repressive complex (PhoRC). PRC2 has 4 constituent proteins: E(z), Su(z)12, Esc, and Pcl in flies and Ezh2, Suz12, Eed, and Phf1 in mammals ( Figure 1A). Ezh2 is the enzymatic component of PRC2, catalyzing methylation of lysine 27 of histone 3 (H3; H3K27), 1-4 whereas Suz12 and Eed are required for complete function of Ezh2 and stable formation of the complex. 5,6 Phf1 influences the enzymatic specificity of Ezh2, promoting trimethylation of H3K27 (H3K27me3) in preference to dimethylation (H3K27me2). 7 Dimethylation is broadly distributed throughout the genome and is thought to have a structural role, whereas H3K27me3 is enriched on promoters and associated with transcriptionally silent regions. [8][9][10][11] There has been a vast expansion in the number of PRC1 components in mammals. PRC1 components include Bmi1 and Mel18 (orthologues of Drosophila Psc), Cbx2, Cbx4, and Cbx8 (orthologues of Drosophila Pc), Scmh1 and Scmh2 (orthologues of Drosophila Scm), Phc1/Rae28, Phc2 and Phc3 (orthologues of Drosophila Ph), and Ring1A and Ring1B (orthologues of Drosophila Ring; Figure 1A). Ring1A and Ring1B are the enzymatic components of PRC1 responsible for the mono-ubiquitination of histone 2A (H2A) at lysine 119 (H2AK119Ub), 12 other components such as Bmi1 and Mel18 are able to stimulate this activity. H2AK119Ub is also associated with transcriptional repression. [13][14][15] PhoRC is composed of the DNA binding transcription factor Yy1 (Drosophila Pho) and Sfmbt1 ( Figure 1A). PhoRC is not known to possess any enzymatic activity. Rather, Sfmbt binds methylated histone residues, 16 and Yy1 is the only mammalian PcG protein with specific DNA binding activity, 17 suggesting that PhoRC may direct PRC1 or PRC2.Some years ago the chromodomain of Drosophila polycomb was shown to bind H3K27me3, leading to the hierarchical recruitment model (reviewed in Wang et al 18 ), in which PRC2 trimethylates H3K27, leading to PRC1 recruitment and ubiquitination of H2AK119. Several lines of evidence support this prevailing model: (1) a high degree of overlap between sites bound by PRC1 and PRC2 in human and mouse cells 9,10,19 ; (2) the failure of Drosophila E(z) mutants to recruit PRC1 20 ; and (3) a correlation between H3K27me3 levels and PRC1 recruitment and H2AK119Ub accumulation. 21 A growing number of reports now suggest that PRC1 and PRC2 may not always act in this fashion: PRC1 binds nucleosomes that lack N-terminal tails in vitro 22 ; PRC1 is recruited in PRC2-deficient mammalian cells 23 ; and genome-wide chromatin immunoprecipitation studies show PRC2 bound to loci devoid of PRC1 19 and vice versa. 15 These observations imply that the interaction between various PRCs is complex.Genome-wide chromatin immunoprecipitation studies suggest PRCs play a role in embryonic stem (ES) cells by repressing key d...

BackgroundSmchd1 is an epigenetic modifier essential for X chromosome inactivation: female embryos lacking Smchd1 fail during midgestational development. Male mice are less affected by Smchd1-loss, with some (but not all) surviving to become fertile adults on the FVB/n genetic background. On other genetic backgrounds, all males lacking Smchd1 die perinatally. This suggests that, in addition to being critical for X inactivation, Smchd1 functions to control the expression of essential autosomal genes.ResultsUsing genome-wide microarray expression profiling and RNA-seq, we have identified additional genes that fail X inactivation in female Smchd1 mutants and have identified autosomal genes in male mice where the normal expression pattern depends upon Smchd1. A subset of genes in the Snrpn imprinted gene cluster show an epigenetic signature and biallelic expression consistent with loss of imprinting in the absence of Smchd1. In addition, single nucleotide polymorphism analysis of expressed genes in the placenta shows that the Igf2r imprinted gene cluster is also disrupted, with Slc22a3 showing biallelic expression in the absence of Smchd1. In both cases, the disruption was not due to loss of the differential methylation that marks the imprint control region, but affected genes remote from this primary imprint controlling element. The clustered protocadherins (Pcdhα, Pcdhβ, and Pcdhγ) also show altered expression levels, suggesting that their unique pattern of random combinatorial monoallelic expression might also be disrupted.ConclusionsSmchd1 has a role in the expression of several autosomal gene clusters that are subject to monoallelic expression, rather than being restricted to functioning uniquely in X inactivation. Our findings, combined with the recent report implicating heterozygous mutations of SMCHD1 as a causal factor in the digenically inherited muscular weakness syndrome facioscapulohumeral muscular dystrophy-2, highlight the potential importance of Smchd1 in the etiology of diverse human diseases.