Double protein knockdown of cIAP1 and CRABP-II using a hybrid molecule consisting of ATRA and IAPs antagonist

Protein knockdown can be achieved by the use of a small molecule that possesses affinity for both the target protein and ubiquitin ligase. We have designed such a degradation-inducing molecule targeting cIAP1 and CRABP-II, which are involved in proliferation of several cancer cell lines and in neuroblastoma growth, respectively. As a CRABP-II-recognizing moiety, all-trans retinoic acid (ATRA, 3), a physiological ligand of CRABP, was chosen. As a cIAP1-recognizing moiety, MV1 (5), which is a cIAP1/cIAP2/XIAP pan-ligand, was chosen. Although cIAP1 itself possesses ubiquitin ligase activity, we expected that its decomposition would be efficiently mediated by related molecules, including cIAP2 and XIAP, which also possess ubiquitin ligase activity. The designed degradation inducer 6, in which ATRA (3) and MV1 (5) moieties are connected via a linker, was synthesized and confirmed to induce efficient degradation of both cIAP1 and CRABP-II. It showed potently inhibited the proliferation of IMR32 cells.     

Small molecules destabilize cIAP1 by activating auto-ubiquitylation

Overexpression of an anti-apoptotic protein cIAP1 caused by its genetic amplification was reported in certain cancers, such as hepatocellular carcinoma, esophageal squamous cell carcinoma, cervical cancer, and lung cancer, which confers resistance to chemotherapy and radiotherapy. Here we report cIAP1 to be selectively down-regulated by a class of small molecules ((-)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-l-leucine methyl ester (ME-BS)), resulting in a sensitization of cancer cells to apoptosis. ME-BS directly interacts with the BIR3 domain of cIAP1, promotes auto-ubiquitylation dependent on its RING domain, and facilitates proteasomal degradation of cIAP1. Other IAPs such as XIAP and cIAP2 were not affected by ME-BS. These results suggest targeted destabilization of cIAP1 by small molecules as a novel method to treat cancers expressing cIAP1, which interferes with treatment. Manipulation of the intrinsic ubiquitin-ligase activity could be a novel strategy to develop small molecules for therapeutic purposes.     

A Ca(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis: possible involvement in the C(4)-phosphoenolpyruvate carboxylase phosphorylation cascade

We have shown previously that fibroblasts derived from fat or dermal tissue differ in their functional properties, such as proliferation rate and contractile properties. To study these differences further, two-dimensional electrophoresis (2D PAGE) was performed on proteins isolated from cultured subcutaneous fat and dermal fibroblasts. The 2D gels were screened for proteins that were differentially expressed in all donors (n = 5). Five protein spots were subjected to further analysis by mass spectrometry. Two proteins could be identified: brain acid soluble protein 1 (BASP1) and cellular retinoic acid binding protein-II (CRABP-II). CRABP-II is of interest in terms of re-epithelialisation and was clearly expressed in dermal fibroblasts but not in fat fibroblasts. Real time PCR was performed to confirm the 2D data on CRABP-II. The CRABP-II mRNA level was significantly increased in dermal tissue and cultured dermal fibroblasts compared to fat tissue and cultured fat-derived fibroblasts, respectively. The mode of action of CRABP-II in skin is to mediate retinoic acid activity. Retinoic acid is known to inhibit migration and to stimulate differentiation of keratinocytes. The expression of CRABP-II by dermal fibroblasts implicates a role for these fibroblasts in wound re-epithelialisation, in contrast to subcutaneous fat-derived fibroblasts.     

Mouse conceptuses have a limited capacity to elevate the mRNA level of cellular retinoid binding proteins in response to teratogenic doses of retinoic acid.

In these studies, we wished to determine the effect of teratogenic doses of retinoic acid on the expression of cellular retinoic acid binding protein I (CRABP-I) mRNA, cellular retinoic acid binding protein II (CRABP-II) mRNA, cellular retinol binding protein I (CRBP-I) mRNA, and cellular retinol binding protein II (CRBP-II) mRNA in mouse conceptuses. Levels of CRABP-II mRNA and CRBP-I mRNA were modestly elevated (2.5-fold and 1.5-fold, respectively) in 9-day gestation conceptuses following treatment of dams with 100 mg/kg b.w. of retinoic acid. These levels were elevated by 6 hr following treatment and remained elevated until 48 and 24 hr, respectively. Two other retinoids, etretinate and retinoyl beta-glucuronide, also moderately elevated CRABP-II mRNA and CRBP-I mRNA levels in conceptuses. In contrast, the levels of CRABP-I mRNA in the conceptuses remained unaffected by treatment with any of these three retinoids. These results demonstrate that conceptuses have a limited capacity to elevate the cellular retinoid binding proteins mRNA levels and presumably the synthesis of their respective proteins in response to high, teratogenic doses of retinoic acid. As a result, an excess of free retinoic acid becomes available to the nuclear retinoic acid receptors, which may lead to inappropriate gene expression and eventual maldevelopment.     

Nuclear shuttling and TRAF2-mediated retention in the cytoplasm regulate the subcellular localization of cIAP1 and cIAP2

Dynamic subcellular localization is an important regulatory mechanism for many proteins. cIAP1 and cIAP2 are two closely related members of inhibitor of apoptosis (IAP) family that play a role both as caspase inhibitors and as mediators of tumor necrosis factor (TNF) receptor signaling. Here, we report that cIAP1 and cIAP2 are nuclear shuttling proteins, whose subcellular localization is mediated by the CRM1-dependent nuclear export pathway. Blocking export with leptomycin B induces accumulation of both endogenous cIAP1 and epitope-tagged cIAP1 and cIAP2 in the nucleus of human cancer cells. We have identified a new CRM1-dependent leucine-rich nuclear export signal (NES) in the linker region between cIAP1 BIR2 and BIR3 repeats. Mutational inactivation of the NES, which is not conserved in cIAP2, reduces cIAP1 nuclear export. Forced relocation of cIAP1 to the nucleus did not significantly alter its ability to prevent apoptosis. Interestingly, co-expression experiments showed that the cIAP1 and cIAP2-interacting protein TNF receptor-associated factor 2 (TRAF2) plays an important role as regulator of IAP nucleocytoplasmic localization, by preventing nuclear translocation of cIAP1 and cIAP2. TRAF2-mediated cytoplasmic retention of cIAP1 was reduced upon TNFalpha treatment. Our results identify molecular mechanisms that contribute to regulate the subcellular localization of cIAP1 and cIAP2. Translocation between different cell compartments may add a further level of control for cIAP1 and cIAP2 activity.     

cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination

The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.     

Molecular determinants of Smac mimetic induced degradation of cIAP1 and cIAP2

The inhibitors of apoptosis (IAP) proteins cIAP1 and cIAP2 have recently emerged as key ubiquitin-E3 ligases regulating innate immunity and cell survival. Much of our knowledge of these IAPs stems from studies using pharmacological inhibitors of IAPs, dubbed Smac mimetics (SMs). Although SMs stimulate auto-ubiquitylation and degradation of cIAPs, little is known about the molecular determinants through which SMs activate the E3 activities of cIAPs. In this study, we find that SM-induced rapid degradation of cIAPs requires binding to tumour necrosis factor (TNF) receptor-associated factor 2 (TRAF2). Moreover, our data reveal an unexpected difference between cIAP1 and cIAP2. Although SM-induced degradation of cIAP1 does not require cIAP2, degradation of cIAP2 critically depends on the presence of cIAP1. In addition, degradation of cIAP2 also requires the ability of the cIAP2 RING finger to dimerise and to bind to E2s. This has important implications because SM-mediated degradation of cIAP1 causes non-canonical activation of NF-κB, which results in the induction of cIAP2 gene expression. In the absence of cIAP1, de novo synthesised cIAP2 is resistant to the SM and suppresses TNFα killing. Furthermore, the cIAP2-MALT1 oncogene, which lacks cIAP2's RING, is resistant to SM treatment. The identification of mechanisms through which cancer cells resist SM treatment will help to improve combination therapies aimed at enhancing treatment response.     

The human anti-apoptotic proteins cIAP1 and cIAP2 bind but do not inhibit caspases

cIAPs (cellular inhibitor of apoptosis proteins) 1 and 2 are able to regulate apoptosis when ectopically expressed in recipient cells and probably also in vivo. Previous work suggested that this is at least partially due to direct caspase inhibition, mediated by two of the three baculovirus IAP repeat (BIR) domains that are contained in these proteins. In support of this we show that the BIR domains 2 and 3 of the two cIAPs are able to bind caspases-7 and -9. However, we demonstrate that neither of these BIR domains is able to inhibit caspases because of critical substitutions in the regions that target caspase inhibition in the X-linked IAP, a tight binding caspase inhibitor. The cIAP BIR domains can be converted to tight binding caspase inhibitors by substituting these critical residues with XIAP residues. Thus, cIAPs maintain protein scaffolds suitable for direct caspase inhibition but have lost or never acquired specific caspase inhibitory interaction sites. Consequently, although the binding function of the cIAP BIRs may be important for their physiologic function, caspase inhibition is not.     

The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination

Human cIAP1 protein is a member of the inhibitor of apoptosis proteins (IAPs) that are involved in apoptosis regulation and an increasing number of other functions, including cell cycle and intracellular signal transduction. In order to identify novel proteins involved in cIAP1 regulation, we performed a yeast two-hybrid screen and identified an F-box protein Fbxo7 as a cIAP1 interacting protein. Co-immunoprecipitation assay showed that cIAP1 can interact with Fbxo7 in human cells. When co-expressed in cells, cIAP1 and Fbxo7 co-localized remarkably both in the cytoplasm and nucleus, and considerable amounts of these often co-localized at one or few distinct Golgi-like structures close to the nucleus. Furthermore, we showed that overexpression of Fbxo7 promotes the ubiquitination of cIAP1. Since F-box proteins are specificity determining subunits of SCF ubiquitin protein ligases, our results suggest that Fbxo7 can mediate the ubiquitination of cIAP1 by SCF ubiquitin protein ligase and thus have important implication in the regulation of cIAP1 function.     

cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.     

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.