Molecular Targeting of Inhibitor of Apoptosis Proteins Based on Small Molecule Mimics of Natural Binding Partners

Kipp, Rachael A.; Case, Martin A.; Wist, Aislyn D.; Cresson, Catherine M.; Carrell, Maria; Griner, Erin; Wiita, Arun P.; Albiniak, Philip A.; Chai, Jijie; Shi, Yigong; Semmelhack, M. F.; McLendon, George

doi:10.1021/bi0121454

Cited by 110 publications

(120 citation statements)

References 21 publications

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“…Other antibodies used were anti-TRAF2 (Santa Cruz, N-19 and C-20), anti-FLAG M2 (Sigma), anti-Myc clone 9E10 (Santa Cruz Biotechnology). AVPI and GVPF peptides were kindly provided by Clemencia Pinilla (Torrey Pines Institute for Medical Studies) and have been described (11,12).…”

Section: Methodsmentioning

confidence: 99%

Distinct BIR Domains of cIAP1 Mediate Binding to and Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 2 and Second Mitochondrial Activator of Caspases

Samuel¹,

Welsh

Lober

et al. 2006

Journal of Biological Chemistry

148

140

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Inhibitor of apoptosis proteins (IAPs) 3 are a family of anti-apoptotic proteins characterized by the presence of baculoviral IAP repeat (BIR) domains (for view, see Ref. 1). IAPs modulate apoptosis by binding and inactivating caspases (2). Several IAPs contain E2 binding RING domains and also operate as E3 ligases that catalyze attachment of ubiquitin to protein substrates. This E3 ligase activity can be directed either toward caspases and caspase regulators or toward signal-transducing proteins primarily involved in activation of NF-B and Jun N-terminal kinase.The human genome includes eight genes that encode BIR-containing proteins. Among these are cIAP1 and cIAP2, which are highly homologous in amino acid sequence and domain structure, containing (from the N to C terminus) three tandem BIR domains followed by a CARD and RING. The cIAP1 and cIAP2 proteins are unique among the BIRfamily proteins in their ability to form complexes with TRAF-family adapter proteins involved in TNF receptor signaling (3-5). These members of the IAP family are known to induce ubiquitination and proteasome-dependent degradation of the TRAFs to which they bind (e.g. TRAF1, TRAF2), but the structural basis for this phenomenon is largely unknown.In addition to binding certain caspases (6), cIAP1 and cIAP2 are reported to bind SMAC, a mitochondrial protein that competes with caspases for binding to IAPs when released into the cytosol (7). In the IAP-family member, XIAP, the binding site for SMAC has been mapped to the third BIR domain (BIR3), where it competes for binding to caspase-9 (8). However, the site on cIAP1 and cIAP2, where SMAC binds has not been previously elucidated, and its relation to the sites required for binding to other targets such as TRAF1 and TRAF2 have not been heretofore defined.Here, we undertook a structure-function analysis of cIAP1 to compare the binding sites for TRAF2 and SMAC and to reveal the functional consequences of disruptions in these binding sites. Our findings indicate that TRAF2 binds the BIR1 domain, whereas SMAC binds the BIR3 domain of cIAP1. The integrity of these binding sites on BIR1 and BIR3 is required for cIAP1-mediated ubiquitination of TRAF2 and SMAC, respectively, revealing the basis for differential targeting of protein substrates by this E3 ligase. We also show evidence of BIR1-dependent modulation of TRAF2-mediated regulation of NF-B. Altogether, these findings demonstrate the modularity and diversification of BIR domains, showing that a single IAP-family protein can direct its E3 ligase activity toward different substrates and can alter the cellular functions of different protein targets in accordance with differences in the specificity of individual BIR domains. MATERIALS AND METHODSPlasmids, Mutagenesis, Recombinant Proteins-A cDNA clone encoding cIAP1 (ID 627116) was obtained from IMAGE consortium through ResGen (Invitrogen) and PCR-subcloned into the pcDNA3-FLAG expression vector. Deletion or point mutations were introduced by PCR or using the QuikChange site-directed mutagen...

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Section: Methodsmentioning

confidence: 99%

Distinct BIR Domains of cIAP1 Mediate Binding to and Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 2 and Second Mitochondrial Activator of Caspases

Samuel¹,

Welsh

Lober

et al. 2006

Journal of Biological Chemistry

148

140

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“…7,8 Thus the strategies that target XIAP synthesis or function are under investigation for the treatment of cancer. 9 Both antisense 10,11 and small molecule inhibitors of XIAP function 12,13 have demonstrated promise in animal models of human cancer.…”

Section: Uiccmentioning

confidence: 99%

Restoration of XAF1 expression induces apoptosis and inhibits tumor growth in gastric cancer

Liston

Cui

et al. 2009

Intl Journal of Cancer

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XAF1 (XIAP-associated factor 1) is a novel XIAP binding protein that can antagonize XIAP and sensitize cells to other cell death triggers. Our previous results have shown that aberrant hypermethylation of the CpG sites in XAF1 promoter is strongly associated with lower expression of XAF1 in gastric cancers. In our study, we investigated the effect of restoration of XAF1 expression on growth of gastric cancers. We found that the restoration of XAF1 expression suppressed anchorage-dependent and -independent growth and increased sensitivity to TRAIL and druginduced apoptosis. Stable cell clones expressing XAF1 exhibited delayed tumor initiation in nude mice. Restoration of XAF1 expression mediated by adenovirus vector greatly increased apoptosis in gastric cancer cell lines in a time-and dose-dependent manner and sensitized cancer cells to TRAIL and drugs-induced apoptosis. Adeno-XAF1 transduction induced cell cycle G2/M arrest and upregulated the expression of p21 and downregulated the expression of cyclin B1 and cdc2. Notably, adeno-XAF1 treatment significantly inhibited tumor growth, strongly enhanced the antitumor activity of TRAIL in a gastric cancer xenograft model in vivo, and significantly prolonged the survival time of animals bearing tumor xenografts. Complete eradication of established tumors was achieved on combined treatment with adeno-XAF1 and TRAIL. Our results document that the restoration of XAF1 inhibits gastric tumorigenesis and tumor growth and that XAF1 is a promising candidate for cancer gene therapy. ' UICCKey words: XAF1; TRAIL; adenovirus; apoptosis; tumorigenesis; cell cycle arrest; gastric cancer; gene therapy The development of cancer is a result of aberrant cellular proliferation and failure of these cells to undergo apoptosis in response to normal signaling cues. 1 Dysregulation of cell death pathways has been termed a 'hallmarkÕ of cancer 2 and is required for tumor initiation, progression and drug resistance. 3,4 Virtually all known apoptosis signal pathways converge on the caspases. 5 The inhibitor of apoptosis (IAP) genes encode a family of proteins that bind and inactivate key caspases involved in the initiation (Caspase-9) and execution (Caspase-3 and -7) of this cascade. 6 An elevated expression level of XIAP, the most potent of the mammalian IAPs, is associated with tumor resistance to cell death cues. 7,8 Thus the strategies that target XIAP synthesis or function are under investigation for the treatment of cancer. 9 Both antisense 10,11 and small molecule inhibitors of XIAP function 12,13 have demonstrated promise in animal models of human cancer.XAF1 has been previously identified as a XIAP binding partner. 13 XAF1 directly interacts with endogenous XIAP and results in XIAP sequestration in nuclear inclusions. XAF1 antagonizes the anticaspase activity of XIAP and reverses the protective effect of XIAP overexpression in cell lines. 13 XAF1 does not contain an amino terminal tetrapeptide motif analogous to Smac/DIABLO or Omi, suggesting that its mechanism of IAP interaction ...

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“…This finding is consistent with specificities obtained with peptides based on the natural IAPbinding protein SMAC, and with a SMAC peptide in which the Val in P2 0 was replaced by Arg. 21 The preference in the third position (P3 0 ) was notably different between BIR2 and BIR3, with the BIR3 domain selecting for Pro, while the BIR2 domain preferred Ala. Arg was the second most preferred residue in P3 0 by BIR3, consistent with Sweeney et al, 7 while Gly was the second most selected for residue in P3 0 by BIR2, consistent with Franklin et al 5 We observed some distinctions in selectivities between the BIR2 and BIR3 domains of XIAP in the fourth position (P4 0 ) in which the former preferred Val and the latter Ile. In summary, the peptide-binding specificity of XIAP-BIR3 (A-I/V-P-I) very closely resembles the mature N-terminal epitope of SMAC (A-V-P-I), while that of XIAP-BIR2 (A-I-A-V) was distinct from this classic consensus ( Figure 3).…”

mentioning

confidence: 99%

The mechanism of peptide-binding specificity of IAP BIR domains

et al. 2008

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We describe the peptide-binding specificity of the baculoviral IAP repeat (BIR) domains of the human inhibitor of apoptosis (IAP) proteins, X-linked IAP, cellular IAP1 and neuronal apoptosis inhibitory protein (NAIP). Synthetic peptide libraries were used to profile each domain, and we distinguish two types of binding specificity, which we refer to as type II and type III BIR domains. Both types have a dominant selectivity for Ala in the first position of the four N-terminal residues of the peptide ligands, which constitute a core recognition motif. Our analysis allows us to define the signature of type III BIRs that demonstrate a preference for Pro in the third residue of the ligand, resembling the classic IAP-binding motif (IBM). The signature of the type II BIRs was similar to type III, but with a striking absence of specificity for Pro in the third position, suggesting that the definition of an IBM must be modified depending on the type of BIR in question. These findings explain how subtle changes in the peptide-binding groove of IAP BIR domains can significantly alter the target protein selectivity. Our analysis allows for prediction of BIR domain protein-binding preferences, provides a context for understanding the mechanism of peptide selection and heightens our knowledge of the specificity of IAP antagonists that are being developed as cancer therapeutics. Cell Death and Differentiation (2008) 15, 920-928; doi:10.1038/cdd.2008 ; published online 1 February 2008Apoptosis is a highly regulated cellular signaling pathway that results in the elimination of unwanted cells from multicellular animals. The apoptotic signaling cascades can be initiated by various extracellular (extrinsic pathway) or intracellular (intrinsic pathway) stimuli and ultimately converge on the activation of a family of proteases known as the caspasesreviewed in Fuentes-Prior and Salvesen. 1 Once active, caspases cleave a limited number of substrates that results in the destruction of the cell and its eventual disposal by phagocytic cells.Members of the inhibitor of apoptosis (IAP) protein family have the unique ability to attenuate apoptosis induced through both intrinsic and extrinsic stimuli. It is well established that the X-linked IAP (XIAP) is capable of blocking apoptosis by direct inhibition of caspase-3, -7, and -9 -reviewed in Salvesen and Duckett 2 and Eckelman et al. 3 The means by which the other members of the IAP family inhibit apoptosis is less understood. Many IAPs have the capacity to bind caspases, yet lack the ability to directly inhibit the proteolytic activity of these enzymes -reviewed in Eckelman et al. 3 The defining feature of the IAP family is the presence of 1-3 baculoviral IAP repeat (BIR) domains. BIRs are small, B70-residue zinc-coordinated domains that have been identified as the regions essential to the IAP-caspase interaction. The BIR domain(s) are necessary for the antiapoptotic activity of most IAPs. Often a surface groove on the BIR domain endows it with an affinity toward extreme N-terminal epitopes...

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Molecular Targeting of Inhibitor of Apoptosis Proteins Based on Small Molecule Mimics of Natural Binding Partners

Cited by 110 publications

References 21 publications

Distinct BIR Domains of cIAP1 Mediate Binding to and Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 2 and Second Mitochondrial Activator of Caspases

Distinct BIR Domains of cIAP1 Mediate Binding to and Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 2 and Second Mitochondrial Activator of Caspases

Restoration of XAF1 expression induces apoptosis and inhibits tumor growth in gastric cancer

The mechanism of peptide-binding specificity of IAP BIR domains

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