Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems

A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.     

Role of XIAP protein, a human member of the inhibitor of apoptosis (IAP) protein family, in phytohemagglutinin-induced apoptosis of human T cell lines

A new family of human genes xiap, hiap-1 and hiap-2, which are homologous to the baculovirus iap (inhibitor of apoptosis) genes cp-iap and op-iap, has been recently cloned and shown to suppress apoptosis after serum withdrawal or exposure to a free radical inducer. In order to examine the role of one of these human genes, namely xiap, in lymphoid cells, we studied XIAP expression, after PHA stimulation in three different human T cell lines. We report here that stimulation with PHA resulted in the human T cell lines undergoing apoptosis, as assessed by DNA fragmentation and by propidiumiodide (PI) staining and flow cytometry. When XIAP protein expression was evaluated by Western blot, we observed that the induction of apoptosis by PHA was associated with a parallel decrease of XIAP expression. We also provide evidence that stably transfected Jurkat cells containing the xiap open reading frame became resistant to PHA-induced apoptosis. These data suggest a role for XIAP protein in the regulation of apoptosis in lymphoid cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Design,synthesis and evaluation of inhibitor of apoptosis protein (IAP) antagonists that are highly selective for the BIR2 domain of XIAP

We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded structure–activity relationship (SAR) studies around these peptidomimetics led to compounds with significantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apoptotic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the biology of XIAP.     

Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori

We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis.     

Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis

BACKGROUND: Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs. Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes besides apoptosis. Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear. RESULTS: We have identified and characterised two Caenorhabditis elegans BIRPs,BIR-1 and BIR-2; these proteins are the only BIRPs in C. elegans. The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos. Overexpression of bir-1 was unable to inhibit developmentally occurring cell death in C. elegans and inhibition of bir-1 expression did not increase cell death. Instead, embryos lacking bir-1 were unable to complete cytokinesis and they became multinucleate. This cytokinesis defect could be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis. CONCLUSIONS: BIR-1, a C. elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis. We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.     

Design,stereoselective synthesis,and biological evaluation of novel tri-cyclic compounds as inhibitor of apoptosis proteins (IAP) antagonists

We recently reported the discovery of octahydropyrrolo[1,2-a]pyrazine A as a lead compound for an inhibitor of apoptosis proteins (IAP) antagonist. To develop IAP antagonists with favorable PK profiles, we designed novel tri-cyclic compounds, octahydro-1H-cyclopropa[4,5]pyrrolo[1,2-a]pyrazines 1 and 2 based on co-crystal structural analysis of A with cellular IAP-1 (cIAP-1). The additional cyclopropane moiety was used to block the predicted metabolic site of compound A without detriment to the binding affinity for cIAP. Compounds 1 and 2 were stereoselectively synthesized via intermediates 4a and 5b′, which were obtained by Simmons?Smith cyclopropanation of ethylester 3a and silyl ether 3b′. Compounds 1 and 2 showed strong growth inhibition in MDA-MB-231 breast cancer cells and improved metabolic stability in comparison to A. Compound 2 exhibited significant in vivo PD effects to increase tumor necrosis factor-alpha mRNA in a dose dependent manner.     

T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis

Identification of T-cell subsets that are infected in vivo is essential to understanding the pathogenesis of human immunodeficiency virus (HIV) disease; however, this goal has been beset with technical challenges. Here, we used polychromatic flow cytometry to sort multiple T-cell subsets to 99.8% purity, followed by quantitative PCR to quantify HIV gag DNA directly ex vivo. We show that resting memory CD4(+) T cells are the predominantly infected cells but that terminally differentiated memory CD4(+) T cells contain 10-fold fewer copies of HIV DNA. Memory CD8(+) T cells can also be infected upon upregulation of CD4; however, this is infrequent and HIV-specific CD8(+) T cells are not infected preferentially. Na?ve CD4(+) T-cell infection is rare and principally confined to those peripheral T cells that have proliferated. Furthermore, the virus is essentially absent from na?ve CD8(+) T cells, suggesting that the thymus is not a major source of HIV-infected T cells in the periphery. These data illuminate the underlying mechanisms that distort T-cell homeostasis in HIV infection.     

Sensitization of human bladder tumor cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis with a small molecule IAP antagonist

Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of bladder tumors are non-muscle invasive at diagnosis, but even after local surgical therapy there is a high rate of local tumor recurrence and progression. Current treatments extend time to recurrence but do not significantly alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein TNF-related apoptosis-inducing ligand (TRAIL) with a small molecule inhibitor of apoptosis proteins (IAP) antagonist to interfere with intracellular regulators of apoptosis in human bladder tumor cells. Our results demonstrate that the IAP antagonist Compound A exhibits high binding affinity to the XIAP BIR3 domain. When Compound A was used at nontoxic concentrations in combination with TRAIL, there was a significant increase in the sensitivity of TRAIL-sensitive and TRAIL-resistant bladder tumor lines to TRAIL-mediated apoptosis. In addition, modulation of TRAIL sensitivity in the TRAIL-resistant bladder tumor cell line T24 with Compound A was reciprocated by XIAP small interfering RNA-mediated suppression of XIAP expression, suggesting the importance of XIAP-mediated resistance to TRAIL in these cells. These results suggest the potential of combining Compound A with TRAIL as an alternative therapy for bladder cancer.     

Biological properties of islets-activating protein (IAP) purified from the culture medium of Bordetella pertussis.

The biological activities were studied of a new protein, islets-activating protein (IAP), purified from the culture medium of Bordetella pertussis. Rats injected intravenously with 1 microgram of purified IAP exhibited markedly enhanced insulin secretory responses to glucose, glucagon, epinephrine, and sulfonylureas over a period from 3 to 10 days after the injection. The degree and duration of the enhancement were proportional to the dose of IAP; the maximal effect induced by 1-2 microgram of IAP persisted for as long as 2 months. There was a highly significant correlation between the enhancement of insulin secretion and suppression of epinephrine hyperglycemia over a wide range of doses of IAP, indicating that suppression of epinephrine hyperglycemia resulted from hypoglycemic action of insulin secreted in response to epinephrine challenge. Additional actions of IAP were observed in mice; mice treated with higher doses of IAP showed symptoms were observed when lower doses of IAP were injected into mice. Thus, it is concluded that IAP is a protein primarily possessing a unique action to potentiate insulin secretory responses of experimental animals to nutritional and hormonal stimuli.     

Immunosuppressive acidic protein (IAP) and CA 125 assays in detection of human ovarian cancer: preliminary results

Serum levels of the immunosuppressive acidic protein (IAP) and CA-125 were measured in 45 patients with ovarian tumors (30 malignant and 15 benign) before surgery. Concentrations of both markers were slightly increased in benign forms but still within the upper limit for controls. The sensitivity of IAP in detecting ovarian cancer was higher than CA-125 (83.4% versus 76.7%). Five false negatives were observed in IAP assay and seven for CA-125. Parallel determination of both markers, however, improved the diagnostic accuracy up to 90.0% of the total malignant cases. Combined measurements of circulating IAP and CA-125 are therefore recommended in the detection of ovarian cancers.     

Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits

Chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone essential for human reproduction. Twenty hCG beta-subunit residues, termed the seatbelt, are wrapped around alpha-subunit loop 2 (alpha 2) and their positions "latched" by a disulfide formed by cysteines at the end of the seatbelt (Cys 110) and in the beta-subunit core (Cys 26). This unique arrangement explains the stability of the heterodimer but raises questions as to how the two subunits combine. The seatbelt is latched in the free beta-subunit. If the seatbelt remained latched during the process of subunit combination, formation of the heterodimer would require alpha 2 and its attached oligosaccharide to be threaded through a small beta-subunit hole. The subunits are known to combine during oxidizing conditions in vitro, and studies described here tested the idea that this requires transient disruption of the latch disulfide, possibly as a consequence of the thioredoxin activity reported in hCG. We observed that alkylating agents did not modify either cysteine in the latch disulfide (Cys 26 or Cys 110) during heterodimer formation in several oxidizing conditions and had minimal influence on these cysteines during combination in the presence of mild reductants (1--3 mM beta-mercaptoethanol). Reducing agents appeared to accelerate subunit combination by disrupting a disulfide (Cys 93--Cys 100) that forms a loop within the seatbelt, thereby increasing the size of the beta-subunit hole. We propose a mechanism for hCG assembly in vitro that depends on movements of alpha 2 and the seatbelt and suggest that the process of glycoprotein hormone subunit combination may be useful for studying the movements of loops during protein folding.     

Islet-activating protein (IAP) reduces bethanechol-stimulated release of pancreatic polypeptide in the dog

The effect of islet-activating protein (IAP) purified from culture medium of Bordetella pertussis was examined in dogs. This was assessed by the levels of pancreatic polypeptide (PP) as well as the responses of plasma insulin and glucagon to a parasympathomimetic agent, bethanechol. Plasma responses of these pancreatic hormones were measured before and 5 days after IAP injection. Although IAP had no significant effect on the bethanechol-stimulated increase in plasma glucose, insulin and glucagon, the PP response to bethanechol was significantly reduced after IAP treatment compared with that before IAP (p less than 0.05). In conclusion, IAP significantly and selectively reduced bethanechol-stimulated PP release in the dog although the mechanism remained to be elucidated.     

Resveratrol-Cu(II) induced DNA breakage in human peripheral lymphocytes: implications for anticancer properties

Resveratrol (3,4',5-trihydroxy stilbene), a plant derived polyphenol found in mulberries, grapes and red wine is considered to possess chemopreventive properties against cancer. It is recognized as a naturally occurring antioxidant but also catalyzes oxidative DNA degradation in vitro in the presence of transition metal ions such as copper. Using a cellular system of lymphocytes isolated from human peripheral blood and Comet assay, we have confirmed that resveratrol-Cu(II) system is indeed capable of causing DNA degradation in cells such as lymphocytes. Also, trans-stilbene, which does not have any hydroxyl groups, is inactive in the lymphocyte system. Pre-incubation of lymphocytes with resveratrol indicates that it is capable of either traversing the cell membrane or binding to it. Our results are in partial support of our hypothesis that anticancer properties of various plant derived polyphenols may involve mobilization of endogenous copper and the consequent prooxidant action.     

Thiol-linked peroxidase activity of human sensitive to apoptosis gene (SAG) protein

SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. Here, we show that SAG possesses a potent peroxidase property to decompose hydrogen peroxide in the presence of dithiothreitol (DTT). However, without DTT as a reducing equivalent, SAG was not able to destroy hydrogen peroxide. The peroxidase activity was completely abolished by the reaction of SAG with N -ethylmaleimide (NEM), a chemical modification agent for the sulfhydryl of proteins. These observations suggested that the sulfhydryl of cysteines in SAG could function as strong nucleophiles to destroy hydrogen peroxide. In addition to the peroxidase activity used to remove hydrogen peroxide, SAG also showed t -butylhydroperoxide ( t -BOOH) and fatty acid hydroperoxide-selective peroxidase activity.     

A highly conserved arginine is critical for the functional folding of inhibitor of apoptosis (IAP) BIR domains

The inhibitor of apoptosis (IAP) proteins are found in all animals and regulate apoptosis (programmed cell death) by binding and inhibiting caspase proteases. This inhibition is overcome by several apoptosis stimulators, including Drosophila Hid and mammalian Smac/DIABLO, which bind to 65-residue baculovirus IAP repeat (BIR) domains found in one to three copies in all IAPs. Virtually all BIRs contain three Cys and a His that bind zinc, a Gly in a tight turn, and an Arg. The functional and structural role of the Arg was investigated in isolated BIR domains from the baculovirus Orgyia pseudotsugata Op-IAP and the Drosophila DIAP1 proteins. Mutation of the Arg to either Ala or Lys abolished Hid and Smac binding to BIRs, despite the Hid/Smac binding site being located on the opposite side of the BIR domain from the Arg. The mutant BIR domains also exhibited weakened zinc binding, increased sensitivity to limited proteolysis, and altered circular dichroism spectra indicative of perturbed domain folding. Examination of known BIR structures indicates that the Arg side chain makes simultaneous bridging hydrogen bonds and a cation-pi interaction for which the Arg guanidino group is uniquely well suited. These interactions are likely critical for stabilizing the tertiary fold of BIR domains in all IAPs, explaining the conservation of this residue.     

Heterogeneous distribution and transmembrane signaling properties of lymphocyte function-associated antigen (LFA-1) in human lymphocyte subsets

The role of LFA-1, a member of the integrin supergene family, in intercellular adhesion, including lymphocyte-endothelial cell (EC) binding, has been established. We now demonstrate that differences in LFA-1 cell surface density are responsible for the variable adhesion efficiency of lymphocyte subsets to EC. Electrophoretic analysis revealed multiple glycosylated isoforms of both alpha and beta subunits, largely as a result of different degrees of sialylation, with variable expression among different lymphocyte subsets. Neuraminidase digestion before EC adhesion increased the binding efficiency of all lymphocyte subsets, although the relative increase in each subset was proportional to the initial LFA-1 sialic acid content. LFA-1 cross-linking resulted in phosphoinositide hydrolysis and a rise in [Ca2+]i when using anti-alpha but not anti-beta subunit antibodies. These findings indicate that the density of LFA-1 on lymphocyte subsets controls their adhesive properties, and that the LFA-1 alpha subunit has transmembrane signaling properties that may result in activation events after interaction with its natural ligands.