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.     

The Oncolytic Activity of Newcastle Disease Virus NDV-HUJ on Chemoresistant Primary Melanoma Cells Is Dependent on the Proapoptotic Activity of the Inhibitor of Apoptosis Protein Livin

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.Newcastle disease virus (NDV) is an avian paramyxovirus that has a potential selective oncolytic effect on human tumors (5, 7, 13, 21, 25, 26). NDV''s natural host is avian, and while mammalian cells bear the sialic acid receptor for NDV and may be infected by the virus, the virus has limited replication capacity in normal mammalian cells (21). We recently reported the development of an attenuated (lentogenic) isolate of NDV (HUJ) that undergoes only one cycle replication in infected mammalian cells (7, 25). NDV-HUJ is a single clone derived from the parental strain NDV Hitchner B1, which contains a mixed viral population. The new virus clone is attenuated due to multiple passages in specific-pathogen-free (SPF) eggs, and its intracerebral pathogenicity index (ICPI) value is low (an ICPI of 0.01 versus an ICPI of 0.93 for the parental NDV Hitchner B1). Sequence analysis of NDV-HUJ indicated 156 changes at the nucleotide sequence level and multiple amino acid changes from the parental B1 virus in all six viral genes (see Fig. S1 in the supplemental material). Although NDV-HUJ is an attenuated virus in chicken, it retains a selective cytotoxic potential for cancer cells, as determined in vitro and in vivo, using murine and human lung carcinomas (25). The oncolytic effect of the virus is apoptosis dependent (25). NDV-HUJ has been applied to treat glioblastoma patients in a phase I/II clinical trials and found to be safe and potentially active (7).The inhibitors of apoptosis proteins (IAPs) are receiving increased attention as key players in the initiation of tumors, their progression, and resistance to chemotherapy treatment (17). To date, eight human IAPs have been identified, including Livin. IAPs are characterized by one or more repeats of a highly conserved 70-amino-acid domain termed the baculovirus IAP repeat (BIR) that can bind and inhibit caspases, some IAPs also contain a conserved sequence termed the RING finger. RING finger proteins might function as E3 ubiquitin ligases; however, the exact nature of the E3 ligase activity of IAPs is still largely unclear.IAPs inhibit apoptosis induced by a variety of stimuli, mainly through their ability to bind and inhibit specific caspases (17). Intense study has shown that the role of IAP in apoptosis regulation is highly diverse, with a prominent role in tumorigenesis and resistance to therapy. Among the human IAPs, XIAP is the best characterized and the most potent caspase inhibitor. The most recently discovered member of this family is Livin, found by us and others (3, 9, 12, 24). Livin contains a single BIR domain and a RING finger (3, 12). We previously found that Livin is specifically cleaved by caspases at the Asp52 residue to produce a large C-terminal fragment, containing both the BIR and the RING domains. After cleavage, truncated Livin (tLivin) acts paradoxically as a proapoptotic factor (18, 19).In the present study we show that chemoresistant melanoma primary cultures that highly express the Livin protein are sensitive to oncolytic NDV-HUJ treatment. This appears to be a result of activation of caspases 8, 3, and 7 that in turn cleave Livin to produce the tLivin. This is a novel regulatory mechanism in which NDV-HUJ can overcome the antiapoptosis function of Livin and expose the “good side” of Livin by inducing the cleavage of Livin to produce the proapoptotic tLivin that subsequently leads to metastatic melanoma cell death.     

Livin promotes Smac/DIABLO degradation by ubiquitin-proteasome pathway

Livin, a member of the inhibitor of apoptosis protein (IAP) family, encodes a protein containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. It has been reported that Livin directly interacts with caspase-3 and -7 in vitro and caspase-9 in vivo via its BIR domain and is negatively regulated by Smac/DIABLO. Nonetheless, the detailed mechanism underlying its antiapoptotic function has not yet been fully characterized. In this report, we provide, for the first time, the evidence that Livin can act as an E3 ubiquitin ligase for targeting the degradation of Smac/DIABLO. Both BIR domain and RING finger domain of Livin are required for this degradation in vitro and in vivo. We also demonstrate that Livin is an unstable protein with a half-life of less than 4 h in living cells. The RING domain of Livin promotes its auto-ubiquitination, whereas the BIR domain is likely to display degradation-inhibitory activity. Mutation in the Livin BIR domain greatly enhances its instability and nullifies its binding to Smac/DIABLO, resulting in a reduced antiapoptosis inhibition. Our findings provide a novel function of Livin: it exhibits E3 ubiquitin ligase activity to degrade the pivotal apoptotic regulator Smac/DIABLO through the ubiquitin-proteasome pathway.     

Three novel Bid proteins generated by alternative splicing of the human Bid gene

Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. Bid(S) contains the N-terminal regulatory domains of Bid without the BH3 domain; Bid(EL) corresponds to full-length Bid with additional N-terminal sequence; and Bid(ES) contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies Bid(EL) induces apoptosis, whereas Bid(S) abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. Bid(ES) induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli.     

Livin, a novel inhibitor of apoptosis protein family member

A novel human inhibitor of apoptosis protein (IAP) family member termed Livin was identified, containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. The mRNA for livin was not detectable by Northern blot in most normal adult tissues with the exception of the placenta, but was present in developmental tissues and in several cancer cell lines. Highest levels were observed in two melanoma-derived cell lines, G361 and SK-Mel29. Transfection of livin in HeLa cells resulted in protection from apoptosis induced by expression of FADD, Bax, RIP, RIP3, and DR6. Similar to other IAP family members, the anti-apoptotic activity of Livin was dependent on the BIR domain. Livin was also capable of inhibiting DEVD-like caspase activity triggered by tumor necrosis factor-alpha. In vitro binding studies demonstrated a direct interaction between Livin and the active form of the downstream caspases, caspase-3 and -7, that was dependent on the BIR domain of Livin. In addition, the unprocessed and cleaved forms of caspase-9 co-immunoprecipitated with Livin in vivo, and recombinant Livin could inhibit the activation of caspase-9 induced by Apaf-1, cytochrome c, and dATP. The subcellular distribution of the transfected Livin was analyzed by immunofluorescence. Both Livin and Survivin were expressed in the nucleus and in a filamentous pattern throughout the cytoplasm. In contrast to the apoptotic activity, the COOH-terminal RING domain mediated its subcellular localization patterning. Further studies found that transfection of an antisense construct against livin could trigger apoptosis specifically in cell lines expressing livin mRNA. This was associated with an increase in DNA fragmentation and in DEVD-like caspase activity. Thus, disruption of Livin may provide a strategy to induce apoptosis in certain cancer cells.     

c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.     

The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase

The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.     

A GH3-like domain in reaper is required for mitochondrial localization and induction of IAP degradation

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. We report here that this domain, although required for IAP destabilization, is not sufficient. Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain was deficient in these properties, these defects could be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells.     

Domain organization of XAF1 and the identification and characterization of XIAP(RING) -binding domain of XAF1

X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) has been implicated as a novel tumor suppressor, which was proposed to exert pro-apoptotic effect by antagonizing the anticaspase activity of XIAP. Here, we delineated the domain architecture of XAF1 by applying limited proteolysis and peptide mass fingerprinting analysis. Our results indicated that XAF1 has a distinct domain organization, with a highly compact N-terminal domain (XAF1(NTD) ) followed by a middle domain (XAF1(MD) ), a 42-residue unstructured linker and a C-terminal domain (XAF1(CTD) ). The search of XIAP binding region within XAF1 revealed that a modest affinity XIAP(RING) binding site (dissociation constant, K(d) , ～18 μM) is located at the C-terminal portion of XAF1. This C-terminal region, embracing XAF1(CTD) and a flexible tail at C-terminus (residue Thr251-Ser301), is functionally identified as XIAP(RING) -binding domain of XAF1 (XAF1(RBD) ) in the present study. We have also mapped the interaction sites for XAF1(RBD) on XIAP(RING) by using NMR spectroscopy. By applying in vitro ubiquitination assay, we observed that XAF1(RBD) /XIAP interaction is essential for the ubiquitination of GST-XAF1(RBD) fusion protein. In addition, the C-terminal XAF1 fragment harboring XAF1(RBD) was found to be substantially ubiquitinated by XIAP(RING) . Base on these observations, we speculate a possible role of XAF1(RBD) in targeting XAF1 for XIAP-mediated ubiquitination.     

RNF152, a novel lysosome localized E3 ligase with pro-apoptotic activities

RING finger protein 152 (RNF152) is a novel RING finger protein and has not been well characterized. We report here that RNF152 is a canonical RING finger protein and has E3 ligase activity. It is polyubiqitinated partly through Lys-48-linked ubiquitin chains in vivo and this phenomenon is dependent on its RING finger domain and transmembrane domain. RNF152 is localized in lysosomes and co-localized with LAMP3, a lysosome marker. Moreover, over-expression of RNF152 in Hela cells induces apoptosis. These results suggest that RNF152 is a lysosome localized E3 ligase with pro-apoptotic activities. It is the first E3 ligase identified so far that is involved in lysosome-related apoptosis.     

A novel human striated muscle RING zinc finger protein, SMRZ, interacts with SMT3b via its RING domain

The RING domain is a conserved zinc finger motif, which serves as a protein-protein interaction interface. Searches of a human heart expressed sequence tag data base for genes encoding the RING domain identified a novel cDNA, named striated muscle RING zinc finger protein (SMRZ). The SMRZ cDNA is 1.9 kilobase pairs in length and encodes a polypeptide of 288 amino acid residues; analysis of the peptide sequence demonstrated an N-terminal RING domain. Fluorescence in situ hybridization localized SMRZ to chromosome 1p33-34. Northern blots demonstrated that SMRZ is expressed exclusively in striated muscle. In the cardiovascular system, SMRZ is more highly expressed in the fetal heart than in the adult heart (slightly higher expression in the ventricle than in the atrium), suggesting that SMRZ is developmentally regulated. SMRZ was found to interact with SMT3b, a ubiquitin-like protein, through the SMRZ-RING domain. This interaction was abolished by mutagenesis of conserved RING domain residues. Transient transfection of SMRZ into C2C12 myoblasts showed localization of SMRZ to the nucleus. These data suggest that SMRZ may play an important role in striated muscle cell embryonic development and perhaps in cell cycle regulation.     

The RING domain of cIAP1 mediates the degradation of RING-bearing inhibitor of apoptosis proteins by distinct pathways

The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR-mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.     

The Ret finger protein induces apoptosis via its RING finger-B box-coiled-coil motif

The Ret finger protein (RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger, a B-box, and a coiled-coil domain (together called RBCC). Although RFP is known to become oncogenic when its RBCC moiety is connected to a tyrosine kinase domain by DNA rearrangement, its biological function is not well defined. Here we show that ectopic expression of RFP in human embryonic kidney 293 cells causes extensive apoptosis, as assessed by multiple criteria. RFP expression activates Jun N-terminal kinase and p38 kinase and also increases caspase-3-like activity. However, RFP failed to release cytochrome c and, therefore, to increase caspase-9-like activity. RFP-induced apoptosis could be blocked by the caspase-8 inhibitor crmA and dominant negative ASK1 but not by Bcl-2. These results reveal a novel RFP death pathway that recruits mitogen-activated protein kinase and caspases independently of mitochondrial events. Domain mapping showed that the intact RBCC moiety is necessary for the pro-apoptotic function of RFP. Moreover, expression of the RBCC moiety further potentiated the pro-apoptotic activity and resulted in a 7-fold increase of caspase activation compared with that induced by full-length RFP. This suggests that a large number of tripartite motif family members sharing the RBCC moiety may participate in the control of cell survival.     

Fold of the conserved DTC domain in Deltex proteins

Human Deltex 3-like (DTX3L) is a member of the Deltex family of proteins. Initially identified as a B-lymphoma and BAL-associated protein, DTX3L is an E3 ligase that regulates subcellular localization of its partner protein, BAL, by a dynamic nucleocytoplasmic trafficking mechanism. Unlike other members of the Deltex family of proteins, DTX3L lacks the highly basic N-terminal motif and the central proline-rich motif present in other Deltex proteins, and instead contains other unique N-terminal domains. The C-terminal domains are, however, homologous with other members of the Deltex family of proteins; these include a RING domain and a previously unidentified C-terminal domain. In this study, we report the high-resolution crystal structure of this previously uncharacterized C-terminal domain of human DTX3L, which we term the Deltex C-terminal domain.     

Fission yeast Dma1 requires RING domain dimerization for its ubiquitin ligase activity and mitotic checkpoint function

In fission yeast (Schizosaccharomyces pombe), the E3 ubiquitin ligase Dma1 delays cytokinesis if chromosomes are not properly attached to the mitotic spindle. Dma1 contains a C-terminal RING domain, and we have found that the Dma1 RING domain forms a stable homodimer. Although the RING domain is required for dimerization, residues in the C-terminal tail are also required to help form or stabilize the dimeric structure because mutation of specific residues in this region disrupts Dma1 dimerization. Further analyses showed that Dma1 dimerization is required for proper localization at spindle pole bodies and the cell division site, E3 ligase activity, and mitotic checkpoint function. Thus, Dma1 forms an obligate dimer via its RING domain, which is essential for efficient transfer of ubiquitin to its substrate(s). This study further supports the mechanistic paradigm that many RING E3 ligases function as RING dimers.     

Cloning and characterization of a novel RING-B-box-coiled-coil protein with apoptotic function

We have identified a novel RING-B-box-coiled-coil (RBCC) protein (MAIR for macrophage-derived apoptosis-inducing RBCC protein) that consists of an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil domain, and a B30.2 domain. MAIR mRNA was expressed widely in mouse tissues and was induced by macrophage colony-stimulating factor in murine peritoneal and bone marrow macrophages. MAIR protein initially showed a granular distribution predominantly in the cytoplasm. The addition of zinc to transfectants containing MAIR cDNA as part of a heavy metal-inducible vector caused apoptosis of the cells characterized by cell fragmentation; a reduction in mitochondrial membrane potential; activation of caspase-7, -8, and -9, but not caspase-3; and DNA degradation. We also found that the RING finger and coiled-coil domains were required for MAIR activity by analysis with deletion mutants.