Baculovirus IAP Repeat Domains Research Articles

Structure of the pre-mRNA leakage 39-kDa protein reveals a single domain of integrated zf-C3HC and Rsm1 modules

In Saccharomyces cerevisiae, the pre-mRNA leakage 39-kDa protein (ScPml39) was reported to retain unspliced pre-mRNA prior to export through nuclear pore complexes (NPCs). Pml39 homologs outside the Saccharomycetaceae family are currently unknown, and mechanistic insight into Pml39 function is lacking. Here we determined the crystal structure of ScPml39 at 2.5 Å resolution to facilitate the discovery of orthologs beyond Saccharomycetaceae, e.g. in Schizosaccharomyces pombe or human. The crystal structure revealed integrated zf-C3HC and Rsm1 modules, which are tightly associated through a hydrophobic interface to form a single domain. Both zf-C3HC and Rsm1 modules belong to the Zn-containing BIR (Baculovirus IAP repeat)-like super family, with key residues of the canonical BIR domain being conserved. Features unique to the Pml39 modules refer to the spacing between the Zn-coordinating residues, giving rise to a substantially tilted helix αC in the zf-C3HC and Rsm1 modules, and an extra helix αAB′ in the Rsm1 module. Conservation of key residues responsible for its distinct features identifies S. pombe Rsm1 and Homo sapiens NIPA/ZC3HC1 as structural orthologs of ScPml39. Based on the recent functional characterization of NIPA/ZC3HC1 as a scaffold protein that stabilizes the nuclear basket of the NPC, our data suggest an analogous function of ScPml39 in S. cerevisiae.

The expanded inhibitor of apoptosis gene family in oysters possesses novel domain architectures and may play diverse roles in apoptosis following immune challenge

BackgroundApoptosis plays important roles in a variety of functions, including immunity and response to environmental stress. The Inhibitor of Apoptosis (IAP) gene family of apoptosis regulators is expanded in molluscs, including eastern, Crassostrea virginica, and Pacific, Crassostrea gigas, oysters. The functional importance of IAP expansion in apoptosis and immunity in oysters remains unknown.ResultsPhylogenetic analysis of IAP genes in 10 molluscs identified lineage specific gene expansion in bivalve species. Greater IAP gene family expansion was observed in C. virginica than C. gigas (69 vs. 40), resulting mainly from tandem duplications. Functional domain analysis of oyster IAP proteins revealed 3 novel Baculoviral IAP Repeat (BIR) domain types and 14 domain architecture types across gene clusters, 4 of which are not present in model organisms. Phylogenetic analysis of bivalve IAPs suggests a complex history of domain loss and gain. Most IAP genes in oysters (76% of C. virginica and 82% of C. gigas), representing all domain architecture types, were expressed in response to immune challenge (Ostreid Herpesvirus OsHV-1, bacterial probionts Phaeobacter inhibens and Bacillus pumilus, several Vibrio spp., pathogenic Aliiroseovarius crassostreae, and protozoan parasite Perkinsus marinus). Patterns of IAP and apoptosis-related differential gene expression differed between the two oyster species, where C. virginica, in general, differentially expressed a unique set of IAP genes in each challenge, while C. gigas differentially expressed an overlapping set of IAP genes across challenges. Apoptosis gene expression patterns clustered mainly by resistance/susceptibility of the oyster host to immune challenge. Weighted Gene Correlation Network Analysis (WGCNA) revealed unique combinations of transcripts for 1 to 12 IAP domain architecture types, including novel types, were significantly co-expressed in response to immune challenge with transcripts in apoptosis-related pathways.ConclusionsUnprecedented diversity characterized by novel BIR domains and protein domain architectures was observed in oyster IAPs. Complex patterns of gene expression of novel and conserved IAPs in response to a variety of ecologically-relevant immune challenges, combined with evidence of direct co-expression of IAP genes with apoptosis-related transcripts, suggests IAP expansion facilitates complex and nuanced regulation of apoptosis and other immune responses in oysters.

CaBir1 functions as an inhibitor-of-apoptosis and affects caspase-like activitiy in Candida albicans

CaMca1 is the only metacaspase in Candida albicans, which shows structural homology to the mammalian caspases. CaMca1 consists of the caspase domain, the P20 and P10 regions, and the conserved catalytic histidine-cysteine dyad that is required for executing apoptosis in C. albicans. However, little is known about the proteolytic processing of CaMca1 or its activation under apoptosis-inducing conditions. To understand the regulation of this process, we characterized CaBir1 which is the single IAP (inhibitor-of-apoptosis protein) in C. albicans. IAPs are a family of proteins whose members all harbor a BIR (baculovirus IAP repeat) domain and negatively regulate apoptosis by inhibiting caspases. We found that the Cabir1/Cabir1 deletion mutant exhibited increased apoptotic phenotypes, such as ROS accumulation, nuclear segmentation, and cell survival, under apoptosis-inducing conditions. Examination of CaMca1 cleavage patterns in response to various apoptotic stresses revealed that these cleavages were stress-specific and dependent on the catalytic histidine-cysteine residues of CaMca1. The Cabir1/Cabir1 mutation was not associated with altered CaMca1 processing with or without apoptotic stimuli, but the Cabir1/Cabir1 mutant exhibited significantly increased caspase-like activities. These results suggest that CaBir1 acts as an apoptosis inhibitor by regulating caspase-like activity, but not CaMca1 processing.

A Spatial and Functional Interaction of a Heterotetramer Survivin–DNA-PKcs Complex in DNA Damage Response

Substantial evidence has shown that overexpression of the inhibitor of apoptosis protein (IAP) Survivin in human tumors correlates significantly with treatment resistance and poor patient prognosis. Survivin serves as a radiation resistance factor that impacts the DNA damage response by interacting with DNA-dependent protein kinase (DNA-PKcs). However, the complexity, molecular determinants and functional consequences of this interrelationship remain largely unknown. By applying co-immunoprecipitation and flow cytometry-based Förster resonance energy transfer assays, we demonstrated a direct involvement of the Survivin baculovirus IAP repeat (BIR) domain in the regulation of radiation survival and DNA repair. This Survivin-mediated activity required an interaction of residues S20 and W67 with the phosphoinositide 3-kinase (PI3K) domain of DNA-PKcs. In silico molecular docking and dynamics simulation analyses, in vitro kinase assays, and large-scale mass spectrometry suggested a heterotetrameric Survivin-DNA-PKcs complex that results in a conformational change within the DNA-PKcs PI3K domain. Overexpression or depletion of Survivin resulted in enhanced PI3K enzymatic activity and detection of differentially abundant phosphopeptides and proteins implicated in the DNA damage response. The Survivin-DNA-PKcs interaction altered the S/T-hydrophobic motif substrate specificity of DNA-PKcs with a predominant usage of S/T-P phosphorylation sites and an increase of DNA-PKcs substrates including Foxo3. These data demonstrate that Survivin differentially regulates DNA-PKcs-dependent radiation survival and DNA double-strand break repair via formation of a Survivin-DNA-PKcs heterotetrameric complex.

Abstract 2939: Canonical and noncanonical survivin isoforms translocate to the plasma membrane and are secreted in exosomes

Abstract Background: Previously we found that survivin is expressed on the surface of cancer cells in vitro, and is targetable using certain survivin-specific antibodies. The survivin gene is alternatively spliced into two alternative isoforms, ΔEX3 and 2B, which diverge structurally from the canonical isoform in their C-termini but retain a conserved BIR (Baculovirus IAP Repeat) domain. Methods: In order to characterize plasma membrane survivin (PM-SVN), tagged proteins representing canonical survivin (isoform 1), Δex3 and 2B were transiently expressed in multiple cancer cell line backgrounds. Relative surface expression was measured by flow cytometry staining of intact cells. Mutant constructs were used to determine the structural requirements for translocation. Imaging flow cytometry (Imagestream) was used to visualize survivin isoform localization, and to detect co-localization on the cell surface. Secreted exosomes were examined for expression of survivin isoforms. Results: Isoforms 1, Δex3 and 2B could be detected on the surface of HEK293T embryonic kidney, HeLa cervical adenocarcinoma, U87 glioblastoma, and A1207 glioblastoma cells. PM-SVN expression was variable, however Δex3 showed robust membrane expression across all cell lines. Deletion of a predicted transmembrane sequence in Δex3's unique C-terminus did not impair its translocation, and deletion of the first 10 amino acids, thought to be required for dimerization, disrupted surface expression only in HEK293T cells. Imaging flow cytometry analysis of cells revealed polar localization of PM-SVN, with specific punctate areas seen in 13-20% of cells. Staining with antibodies targeting both isoform 1 and Δex3 showed co-immunofluorescence in cell membrane puncta, indicating the formation of heterodimers. Stressing cells by heat shock increased the proportion of PM-SVN in 293T and A1207 cells; in 293T, this was accompanied by enrichment of survivin protein in secreted exosomes. Conclusions: In addition to canonical survivin, isoforms Δex3 and 2B are displayed on the surface of cancer cells, and can be secreted as exosomal cargo. Membrane translocation may be stress-induced and appears to require the conserved BIR domain. PM-SVN shows a polar distribution on the cell membrane, and isoforms 1 and Δex3 heterodimerize within these puncta. Citation Format: Sheila Figel, Meaghan Birkemeier, Sanam Dharma, Emma Steinmetz, Michael Ciesielski, Robert Fenstermaker. Canonical and noncanonical survivin isoforms translocate to the plasma membrane and are secreted in exosomes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2939.

Third BIR domain of XIAP binds to both Cu(II) and Cu(I) in multiple sites and with diverse affinities characterized at atomic resolution

The X-chromosome linked inhibitor of apoptosis, XIAP, is mainly known as the inhibitor of caspases by direct interaction with caspases with its baculoviral IAP repeat (BIR) domains. XIAP has three BIR domains and each BIR domain contains a zinc binding site, normally known as zinc finger motif. Recent studies showed that XIAP is involved in copper homeostasis in cells and the BIR domains bind copper ion. However, structural details of the second and third BIR domain, BIR2 and BIR3, in XIAP, with copper as well as the binding modes are not known. In the present work we characterize the structural properties of BIR3 in solution by high resolution NMR and other biophysical techniques. The interaction of BIR3 with copper both in vitro and in cell lysates was analyzed. Our results show that BIR3 is able to form stable complexes both with Cu(II) and Cu(I), whereas zinc binding site is not affected and zinc retains tightly bound in the zinc finger during these interactions. Surprisingly, BIR3 has multiple binding sites for Cu(II) and Cu(I) but with varied binding affinities. In addition, the solvent exposed Cys351 is readily oxidized by Cu(II) resulting an intermolecular disulfide bond either between two BIR3 molecules or a mixed disulfide bond with glutathione in cell lysates.

Dissecting the Function of IAP (Inhibitor of Apoptosis) Protein Domains in Inhibiting an Apoptotic Caspase

Apoptosis (programmed cell death) is an essential process in all animals for normal development and tissue regeneration. Apoptosis also eliminates cells at risk for becoming cancerous or spreading a viral infection. Apoptosis requires the activity of intracellular caspase enzymes that are normally kept inhibited in living cells by inhibitor of apoptosis proteins (IAPs). Certain apoptosis‐promoting proteins bind IAPs to relieve caspase inhibition. IAPs contain 2–3 BIR (baculovirus IAP repeat) domains with intervening linkers. In many IAPs, residues in a linker bind and block the caspase active site but the BIR domains also influence caspase inhibition. To determine how the BIRs contribute to caspase inhibition, we compared the ability of different fragments of the Drosophila IAP DIAP1 to inhibit a Drosophila caspase using purified recombinant proteins and an in vitro caspase assay. 2 μM of a DIAP1 fragment containing the BIR1 and BIR2 domains with intervening linker inhibited the caspase by 72 ± 1%. Removing either BIR domain reduced the inhibition to 39–45 ± 10%. Neither BIR domain alone inhibited the caspase even at 6 μM. However, 6 μM added BIR1 domain interfered with the 2 μM BIR1‐linker, reducing its caspase inhibition from 56 ± 1% to 30 ± 2%. An affinity chromatography binding assay detected direct BIR1 domain binding to the caspase. This suggests that the BIR1 interfered with the BIR1‐linker by binding to and blocking a critical BIR1‐binding site on the caspase. Thus, the BIR1 likely functions within DIAP1 by helping anchor the linker to the caspase active site. The BIR2 domain did not interfere with the linker‐BIR2 nor show direct binding to the caspase, indicating that its mechanism for enhancing caspase inhibition by DIAP1 must differ from that of the BIR1 domain. The role of the BIR2 as well as of other linker residues is being further investigated.Support or Funding InformationThis research was funded by a Kutztown University Research Grant to M. Junker and a Kutztown University BEARS grant and a Kutztown University Undergraduate Research Committee Grant to M. Storm.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 4918: Is Livin a protagonist of mucinous adenocarcinoma histology in colorectal cancer

Abstract The mechanism of cancer resistance to chemotherapy regimen remains uncertain. Colorectal mucinous adenocarcinoma is one of the distinct histological subtypes of the disease implicated in chemotherapeutic resistance associated with nodal and peritoneal metastases and worse disease-free survival as an index of poor prognosis. One of the important acquired capabilities used by the cancer cells to resist anticancer therapies is evasion of apoptosis possibly via inhibitor of apoptosis proteins for which Livin is one. This protein contains baculoviral IAP repeat domains in addition to a RING finger, a protein-protein motif important for binding and inhibition of active caspases that interfere with intrinsic and/or extrinsic pathway and the ensuing blockade of apoptosis. Thus, understanding the molecular events on how cancer cells of mucinous histology evade apoptotic death may provide a novel paradigm for a molecular targeted therapy in the management of colorectal mucinous adenocarcinoma. In this study, the expression pattern and prognostic value of IAP family protein, Livin, in colorectal cancer patients with mucinous histology pre and post-chemotherapy regimen was investigated. Tissue sections from advanced stage colorectal cancer patients who were treated or untreated with neo-adjuvant FOLFOX chemotherapy before curative resection were included in this study. The tissue sections were grouped according to colorectal adenocarcinomas showing mucinous histology and non-mucinous component. Histological study including Haematoxylin and Eosin, and immunohistochemistry for Anti-Livin and DNA mismatched repair proteins were carried out. Immunofluorescence study was performed to clarify the expression pattern of the protein using Anti-Livin antibody. Protein expression quantification study was also used. Results show significant cytoplasmic localisation and expression of Livin protein in the colorectal cancer cells. The Livin protein expression was found to be increased by more than a one-fold post-chemotherapy treatment when compared with pre-chemotherapy treated patients with mucinous histology of colorectal cancer. Younger patients were found to have a greater probability of colorectal mucinous adenocarcinoma diagnosis and worse prognosis. Our findings show that Livin-induced inhibition of apoptosis activity can be a target for novel approaches to treatment and prevention of chemotherapy associated drug resistance in mucinous histology colorectal cancer since Livin overexpression has been associated with metastases and worse disease-free survival. More study is needed Citation Format: Mohammed Faruk, Abdulmumini Hassan Rafindadi, Sani Ibrahim, Surajo Mohammed Aminu, Surajo Mohammed Aminu, Ahmed Adamu, Ahmed Adamu, Yawale Iliyasu, Adamu Abdullahi, Mohammed Sani Shehu, Abdullahi Mohammed, John Idoko, Abdullahi Jibril Randawa, Abdullahi Jibril Randawa, Atara Ntekim, Saad Aliyu Ahmed, Aishatu Suleiman Maude, Almustapha Aliyu Liman, Abubakar Sani, Khalid Zahir Shah, Yahaya Ukwenya, Yahaya Ukwenya, Cheh Augustine Awasum, Kasimu Umar Adoke, James Olowu Enemari, James Olowu Enemari, James Olowu Enemari, Andrew Jonathan Nok. Is Livin a protagonist of mucinous adenocarcinoma histology in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4918. doi:10.1158/1538-7445.AM2017-4918

Molecular cloning and characterization of an inhibitor of apoptosis protein in the mud crab Scylla paramamosain: its potential anti-apoptosis role under air exposure stress

The mud crab Scylla paramamosain is a widely farmed commercial species in southeast coastal areas of China. The crabs are placed in water-free containers for transportation to inland markets, thus are exposed to dry conditions for more than 72 h after capture and may suffer from air exposure stress, triggering cell apoptosis leading to death. To evaluate whether an inhibitor of apoptosis protein (IAP) is involved in apoptosis resistance against air exposure stress in crustaceans, SpIAP was cloned and investigated for the first time. The full length of SpIAP was 3351 bp, encoding a polypeptide of 662 amino acids. The predicted SpIAP protein contained three baculoviral IAP repeat domains and one really interesting new gene (RING) finger domain. Protein basic local alignment search tool and phylogenetic analysis results showed that SpIAP was clustered together with other crustaceans IAPs. SpIAP was detected in all the examined tissues and predominantly expressed in the hepatopancreas. When crabs were challenged with air exposure for 12 h, the expression level of SpIAP in the hepatopancreas was significantly increased in the experimental group compared with the control group. A RNA interference assay and flow cytometry analysis showed that when SpIAP was silenced, the cell apoptotic rate significantly increased after 24 h air exposure. These results suggested that SpIAP was involved in an anti-apoptosis response induced by air exposure in S. paramamosain.

Inducing death in tumor cells: roles of the inhibitor of apoptosis proteins.

The heterogeneous group of diseases collectively termed cancer results not just from aberrant cellular proliferation but also from a lack of accompanying homeostatic cell death. Indeed, cancer cells regularly acquire resistance to programmed cell death, or apoptosis, which not only supports cancer progression but also leads to resistance to therapeutic agents. Thus, various approaches have been undertaken in order to induce apoptosis in tumor cells for therapeutic purposes. Here, we will focus our discussion on agents that directly affect the apoptotic machinery itself rather than on drugs that induce apoptosis in tumor cells indirectly, such as by DNA damage or kinase dependency inhibition. As the roles of the Bcl-2 family have been extensively studied and reviewed recently, we will focus in this review specifically on the inhibitor of apoptosis protein (IAP) family. IAPs are a disparate group of proteins that all contain a baculovirus IAP repeat domain, which is important for the inhibition of apoptosis in some, but not all, family members. We describe each of the family members with respect to their structural and functional similarities and differences and their respective roles in cancer. Finally, we also review the current state of IAPs as targets for anti-cancer therapeutics and discuss the current clinical state of IAP antagonists.

The Activator of Apoptosis Smac-DIABLO Acts as a Tetramer in Solution

Smac-DIABLO in its mature form (20.8 kDa) binds to baculoviral IAP repeat (BIR) domains of inhibitor of apoptosis proteins (IAPs) releasing their inhibitory effects on caspases, thus promoting cell death. Despite its apparent molecular mass (∼100 kDa), Smac-DIABLO was held to be a dimer in solution, simultaneously targeting two distinct BIR domains. We report an extensive biophysical characterization of the protein alone and in complex with the X-linked IAP (XIAP)-BIR2-BIR3 domains. Our data show that Smac-DIABLO adopts a tetrameric assembly in solution and that the tetramer is able to bind two BIR2-BIR3 pairs of domains. Our small-angle x-ray scattering-based tetrameric model of Smac-DIABLO/BIR2-BIR3 highlights some conformational freedom of the complex that may be related to optimization of IAPs binding.

The BIR and BIR-like domains of Bombyx mori nucleopolyhedrovirus IAP2 protein are required for efficient viral propagation

The baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) possesses two genes, iap1 and iap2, which encode inhibitor of apoptosis (IAP) proteins. We previously showed that although both genes are dispensable for viral propagation, iap2 is required for efficient viral propagation in cultured cells. BmNPV IAP2 contains three putative functional domains: a baculovirus IAP repeat (BIR), a BIR-like (BIRL) domain, and a RING finger domain. To identify the domain affecting viral growth, we generated a series of BmNPV bacmids expressing iap2 derivatives lacking one or two domains, or possessing a single amino acid substitution to abolish IAP2 ubiquitin ligase activity. We examined their properties in both cultured cells and B. mori larvae. We found that either the BIR or BIRL domain of IAP2 plays an important role in BmNPV infection, and that the RING finger domain, which is required for ubiquitin ligase activity, does not greatly contribute to BmNPV propagation. This is the first study to identify functional domains of the baculovirus IAP2 protein.

Targeting inhibitor of apoptosis proteins for cancer therapy: a double-edge sword?

Evasion of programmed cell death is a hallmark of human cancers. Inhibitor of apoptosis (IAP) proteins such as X-chromosome– linked IAP (XIAP) and cellular IAP (cIAP) proteins play an important role in supporting cell survival by blocking cell death. IAP proteins comprise at least one of the signature baculoviral IAP repeat domains, a protein-protein motif critical for their binding to and inhibition of caspases to block the implementation of cell death. Some IAP proteins also contain the really interesting new gene domain with E3 ubiquitin ligase activity responsible for ubiquitination, leading to proteasomal degradation or altered signaling functions of substrates. Because IAP proteins are expressed at high levels in human cancers and contribute to tumor progression, treatment resistance, and poor prognosis, they have attracted considerable attention as therapeutic targets for drug development. To this end, smallmolecule inhibitors have been developed that mimic the N-terminal portion of second mitochondria-derived activator of caspases (Smac), an endogenous IAP antagonist that is released from mitochondria into the cytosol during apoptosis. IAP inhibitors, also called Smac mimetics, are composed of one (monovalent) or two (bivalent) Smac-mimicking units. Smac mimetics induce apoptosis in cancer cells by binding to and neutralizing XIAP, thereby releasing caspases from the inhibitory functions of XIAP (Fig 1). In addition, the binding of Smac mimetics to cIAP proteins stimulates the E3 ligase activity of cIAP1 and cIAP2, leading to their autoubiquitination and degradation via the proteasome (Fig 1). Because cIAP proteins constitutively mediate ubiquitination and degradation of nuclear factor kappa B (NFB) –inducing kinase, a key component of the noncanonical NFB pathway, the Smac mimetic–mediated depletion of cIAP proteins results in accumulation of NFB–inducing kinase, activation of the noncanonical NFB pathway, and upregulation of NFB target genes, including inflammatory cytokines such as tumor necrosis factor (TNF ). TNF , a member of the death receptor ligand family, then triggers cell death on binding to its cognate cell surface receptor TNF receptor 1 in an autocrine/paracrine manner. The concomitant Smac mimetic– triggered loss of cIAP proteins is critical for TNF -induced cell death by shutting off ubiquitination of receptor-interacting protein 1, thereby promoting its interaction with Fas-associated protein with death domain and caspase-8 to form a cytosolic cell death complex that drives caspase-8 activation and apoptosis. Smac mimetics have repeatedly been shown to either directly induce apoptosis or to sensitize cancer cells for apoptosis in response to additional cytotoxic treatments. More than a decade ago, a proof-of-concept study used a preclinical in vivo cancer model to demonstrate the feasibility of targeting IAP proteins by Smac mimetics for cancer therapy. Currently, five distinct Smac mimetic compounds have been evaluated in early clinical trials, including LCL161, a monovalent, orally bioavailable drug. The article by Infante et al that accompanies this Understanding the Pathway reports on the first-inhuman, phase I dose-escalation study of LCL161 in patients with advanced solid tumors. Objectives of the study were to determine the maximum-tolerated dose, dose-limiting toxicities (DLTs), TNFα

X-linked inhibitor of apoptosis protein (XIAP) lacking RING domain localizes to the nuclear and promotes cancer cell anchorage-independent growth by targeting the E2F1/Cyclin E axis.

The inhibitor of apoptosis protein XIAP (X-linked inhibitor of apoptosis protein) is a well-documented protein that is located in cytoplasm acting as a potent regulator of cell apoptosis. Here, we showed that expressing XIAP with RING (Really Interesting New Gene) domain deletion (XIAP△RING) in cancer cells promoted cancer cell anchorage-independent growth and G1/S phase transition companied with increasing cyclin e transcription activity and protein expression. Further studies revealed that XIAP△RING was mainly localized in nuclear with increased binding with E2F1, whereas XIAP with BIR (Baculoviral IAP Repeat) domains deletion (XIAP△BIRs) was entirely presented in cytoplasma with losing its binding with E2F1, suggesting that RING domain was able to inhibit BIR domains nuclear localization, by which impaired BIRs binding with E2F1 in cellular nucleus in intact cells. These studies identified a new function of XIAP protein in cellular nucleus is to regulate E2F1 transcriptional activity by binding with E2F1 in cancer cells. Our current finding of an effect of XIAP△RING expression on cancer cell anchorage-independent growth suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to its impact on cellular apoptosis.

Caspase-dependent regulation of the ubiquitin–proteasome system through direct substrate targeting

Drosophila inhibitor of apoptosis (IAP) 1 (DIAP1) is an E3 ubiquitin ligase that regulates apoptosis in flies, in large part through direct inhibition and/or ubiquitinylation of caspases. IAP antagonists, such as Reaper, Hid, and Grim, are thought to induce cell death by displacing active caspases from baculovirus IAP repeat domains in DIAP1, but can themselves become targets of DIAP1-mediated ubiquitinylation. Herein, we demonstrate that Grim self-associates in cells and is ubiquitinylated by DIAP1 at Lys136 in an UbcD1-dependent manner, resulting in its rapid turnover. K48-linked ubiquitin chains are added almost exclusively to BIR2-bound Grim as a result of its structural proximity to DIAP1's RING domain. However, active caspases can simultaneously cleave Grim at Asp132, removing the lysine necessary for ubiquitinylation as well as any existing ubiquitin conjugates. Cleavage therefore enhances the stability of Grim and initiates a feed-forward caspase amplification loop, resulting in greater cell death. In summary, Grim is a caspase substrate whose cleavage promotes apoptosis by limiting, in a target-specific fashion, its ubiquitinylation and turnover by the proteasome.

Polypeptide chain release factor eRF3 is a novel molecular partner of survivin

The eukaryotic class II polypeptide chain release factor (eRF3) is an eRF1- and ribosome-dependent GTPase involved in translation termination of protein biosynthesis. eRF3 is a multifunctional protein that is also involved in chromosomal segregation and cytokinesis during mitosis. Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is involved in the organisation of spindle and cell apoptosis. Interaction between survivin and eRF3a-F3 or eRF3b, encoded by the GSPT1 and GSPT2 genes, respectively, was confirmed using yeast two-hybrid (Y2H) and pull-down assays in vitro, and co-immunoprecipitation in vivo. The domains involved in the formation of the survivin-eRF3s complex have been identified. The sites on survivin that interact with eRF3 are located in the baculovirus IAP repeat domain (residues 65-76), which forms a beta-strand structure with an overall negative charge. The sites on eRF3 that interact with survivin were localised to the N-terminal domain(NTD; residues 131-200). Cell localisation experiments indicate that both factors are in the nucleus, suggesting that they cooperatively function in nuclear processes.

Immunotherapeutic benefit of α‐interferon (IFNα) in survivin2B‐derived peptide vaccination for advanced pancreatic cancer patients

Survivin, a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain, is highly expressed in cancerous tissues but not in normal counterparts. Our group identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), that is recognized by CD8 + CTLs and functions as an immunogenic molecule in patients with cancers of various histological origins such as colon, breast, lung, oral, and urogenital malignancies. Subsequent clinical trials with this epitope peptide alone resulted in clinical and immunological responses. However, these were not strong enough for routine clinical use as a therapeutic cancer vaccine, and our previous study of colon cancer patients indicated that treatment with a vaccination protocol of survivin-2B80-88 plus incomplete Freund's adjuvant (IFA) and α-interferon (IFNα) conferred overt clinical improvement and enhanced the immunological responses of patients. In the current study, we further investigated whether this vaccination protocol could efficiently provide not only improved immune responses but also better clinical outcomes for advanced pancreatic cancers. Tetramer and enzyme-linked immunosorbent spot analysis data indicated that more than 50% of the patients had positive clinical and immunological responses. In contrast, assessment of treatment with IFNα only to another group of cancer patients resulted in no obvious increase in the frequency of survivin-2B80-88 peptide-specific CTLs. Taken together, our data clearly indicate that a vaccination protocol of survivin-2B80-88 plus IFA and IFNα is very effective and useful in immunotherapy for this type of poor-prognosis neoplasm. This trial was registered with the UMIN Clinical Trials Registry, no. UMIN000000905.

Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic

Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.

Characterization of ML-IAP protein stability and physiological role in vivo

ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.

Role of Melanoma Inhibitor of Apoptosis (ML-IAP) Protein, a Member of the Baculoviral IAP Repeat (BIR) Domain Family, in the Regulation of C-RAF Kinase and Cell Migration

Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration.