Among the most promising chemopreventive agents, certain natural polyphenols have recently received a great deal of attention because of their demonstrated inhibitory activity against tumorigenesis. In view of their anticancer properties, these compounds also hold great promise as potential chemotherapeutic agents. However, to translate these chemopreventive agents into chemotherapeutic compounds, their exact mechanisms of action must be delineated. By using a multidisciplinary approach guided by modern nuclear magnetic resonance spectroscopy techniques, fluorescence polarization displacement assays, and cell-based assays, we have begun to unravel the mechanisms of actions of certain polyphenols such as Gossypol (a compound from cotton seed extracts) and Purpurogallin (a natural compound extracted from Quercus sp. nutgall) and their derivatives. Our findings suggest that these natural products bind and antagonize the antiapoptotic effects of B-cell lymphocyte/leukemia-2 (Bcl-2) family proteins such as Bcl-x(L). Our in vitro and in vivo data not only open a window of opportunities for the development of novel cancer treatments with these compounds but also provide structural information that can be used for the design and development of novel and more effective analogues.
The functional importance of APE1 nucleolar accumulation is described. It is shown that acetylation of Lys27–35, affecting local conformation, regulates APE1 function by 1) controlling its interaction with NPM1 and rRNA and its nucleolar accumulation, 2) modulating K6/K7 acetylation status, and 3) promoting APE1 BER activity in cells.
Sterile alpha motif (Sam) domains are protein interaction modules that are implicated in many biological processes mainly via homo-and hetero-dimerization. It has been recently reported that the lipid phosphatase Ship2 regulates endocytosis of the EphA2 receptor, a process that has been investigated as a possible route to reduce tumor malignancy. A heterotypic Sam-Sam domain interaction is mediating this process. Here, we report NMR and ITC (Isothermal Titration Calorimetry) studies on the Sam domain of Ship2 revealing its three-dimensional structure and its possible mode of interaction with the Sam domain from the EphA2 receptor. These studies have also resulted in the identification of a minimal peptide region of Ship2 that retains binding affinity for the Sam domain of EphA2 receptor. Hence, this peptide and the detection of key structural elements important for EphA2 receptor endocytosis provide possible ways for the development of novel small molecule antagonists with potential anti-cancer activity.The Src homology 2 domain-containing phosphoinositide-5-phosphatase 2 (Ship2) catalyzes the conversion of phosphatidylinositol(3,4,5)P3 (PtIns(3,4,5)P3) to phosphatidylinositol(3,4) P2 (PtIns(3,4)P2), thus inhibiting processes that are activated by the phospatidylinositol 3 kinase (PI3K) (1,2) ( Figure 1). Specifically, Ship2 plays a role in insulin resistance and obesity by interfering with the phosphoinositide-dependent kinase 1 (PDK-1) activation (3,4) and hence, it has been proposed as a potential target in drug discovery for type 2 diabetes (5). Less clear is the role of Ship2 in cancer; however, very recently a novel function for the protein has emerged as regulator of the Ephrin A2 (EphA2) receptor endocytosis (6). Since the EphA2 receptor is over-expressed in a variety of cancers (i.e: colon, lung, breast and prostate cancers) (7,8), the processes of its endocytosis and the consequent degradation have been investigated for their potential correlation to decreased tumor malignancy (8,9). In vitro studies, have demonstrated that Ship2 over-expression in malignant breast cancer cells reduces EphA2 receptor endocytosis while decreased levels of Ship2 facilitate receptor internalization and subsequent degradation (6), and that a heterotypic Sam-Sam domain interaction is needed to engage Ship2 to the EphA2 receptor site (6) (Figure 1 MATERIALS AND METHODS Protein expressionShip2-Sam and EphA2-Sam were expressed as recombinant proteins in E. coli. Synthetic genes coding for residues from 1194 to 1258 of human Ship2 (UniprotKB/TrEMBL code: O15357) and encompassing the Sam domain (residues from 1196 to 1258) were purchased from Retrogen (San Diego, CA). Genes were cloned into the PET15b plasmid and transformed using BL21-Gold (DE3) competent cells (Stratagene).The PET15b plasmid carrying synthetic genes coding for residues from 901 to 976 of the human EphA2 receptor (Swiss-Prot/TrEMBL: P29317) and encompassing the Sam domain (residues from 904 to 968) was purchased from Celtek (Nashville, TN). PET15b plas...
Antiapoptotic Bcl-2-family proteins Bcl-2 and Bcl-X(L) have been recently validated as drug discovery targets for cancer. Here, by using a combination of molecular modeling, NMR-based structural analysis, fluorescence polarization assays, and cell-based assays, we have designed and characterized a novel proapoptotic compound targeting these proteins. Our compound, Apogossypol, is capable of binding and inhibiting Bcl-2 and Bcl-X(L) with high affinity and induces apoptosis of tumor cell lines. Mechanistic studies on the action of our compound were also performed via confocal microscopy that provided real-time detection of the interaction with Bcl-X(L) in intact cells. Finally, preliminary data on cells freshly isolated from patients affected by chronic lymphocytic leukemia strongly suggest potential applications of Bcl-2 antagonists as chemosensitizers in cancer therapy.
Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets ␤-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating ␤-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.Polyubiquitination of specific proteins in cells involves the concerted action of E1, 5 E2, and E3 enzymes. First, E1 covalently binds and activates ubiquitin for subsequent transfer to one of several E2s. The latter can in turn directly transfer its bound ubiquitin to the amino groups of lysine side chains in target proteins. More often, however, E3 ligases recognize substrates and direct their interaction with E2s, resulting in the highly specific regulation of target protein polyubiquitination (1, 2). Humans carry two highly related genes, siah1 and siah2 (3), that encode the mammalian homologs of the Drosophila Sina protein, which is required for R7 photoreceptor cell differentiation within the sevenless pathway (4, 5). Sina/Siah proteins are E3 ligases, acting either as single proteins or as part of a multiprotein complex that is analogous to the Skp1-cullin-1-F-box (SCF) complex. Among the targets of Sina/Siah are NcoR (6), DCC (7), c-Myb (8), BOB-1/OBF-1 (9, 10), Peg3/Pw1 (11), Kid (12), Numb (13) (24), and ␣-ketoglutarate dehydrogenase (25). In addition, Siah interacts with adenomatous polyposis coli, a tumor suppressor involved in colon cancers (26); VAV, a nucleotide exchange factor involved in control of Rho/Rac proteins (27); BAG-1, a Hsp70/Hsc70-binding protein that modulates pathways involved in the control of cell proliferation, death, and migration (28, 29); and Dab-1, an inhibitor of Siah1 (30). However, Sina/Siah does not appear to target phyllopod, adenomatous polyposis coli, VAV, BAG-1, or Dab-1 for polyubiquitination and degradation. Thus, not all Siahbinding proteins are targets of Siah-mediated degradation.Recently, we discovered a novel pathway for ␤-catenin degradation involving a complex formed by Siah1, SIP, the adaptor protein Skp1 that is common to the SCF complex, and the F-box protein Ebi that binds ␤-catenin independent of phosphorylation (31). Siah1 expression is upregulated by p53, revealing a link between genotoxic injury and destruction of ␤-catenin, reduced Tcf/LEF activity, and cell cycle arrest (31). Siah1 is a dimeric protein that contains an N-terminal RING domain (an E2 binding domain) followed by...
Bcl-2 family proteins play a crucial role in tissue homeostasis and apoptosis (programmed cell death). Bid is a proapoptotic member of the Bcl-2 family, promoting cell death when activated by caspase-8. Following an NMR-based approach (structure-activity relationships by interligand NOE) we were able to identify two chemical fragments that bind on the surface of Bid. Covalent linkage of the two fragments led to high-affinity bidentate derivatives. In vitro and in-cell assays demonstrate that the compounds prevent tBid translocation to the mitochondrial membrane and the subsequent release of proapoptotic stimuli and inhibit neuronal apoptosis in the low micromolar range. Therefore, by using a rational chemical-biology approach, we derived antiapoptotic compounds that may have a therapeutic potential for disorders associated with Bid activation, e.g., neurodegenerative diseases, cerebral ischemia, or brain trauma.apoptosis ͉ drug discovery ͉ neurodegeneration P rogrammed cell death (1-3) is a process associated with several pathologies such as neurodegenerative diseases, spinal cord injury, amyotrophic lateral sclerosis, and brain ischemia (4-6). Bid (7, 8) is a proapoptotic member of the Bcl-2 family proteins, and its altered expression accounts for onset and propagation of these pathologies (4-6). The sequence of events leading to programmed cell death has been well characterized in nonneuronal cells (1-3), where Bid provides one mechanism by which TNF-Fas family death receptor activation is linked to downstream events (9). These death receptors activate caspase-8, which cleaves Bid to its truncated active form, tBid (8). tBID targets the outer mitochondrial membrane and induces conformational changes in Bak and Bax (10), which results in the release of proapoptotic stimuli such as Smac and cytochrome c (11). Cytochrome c, together with APAF-1 and caspase-9, form the apoptosome complex, which results in the activation of caspase-3 and other effector caspases, which ultimately cause cell death (12).Recent studies clearly point to Bid as a mediator upstream of mitochondria in neuronal death after cerebral ischemia (13). By using an in vivo model of mild focal cerebral ischemia and an in vitro neuronal oxygen glucose deprivation (OGD) that favors apoptotic cell death, we showed that Bid is a critical mediator of ischemic cell death within the CNS (13). Reduced cell death was observed in highly enriched neuronal cultures from BidϪ͞Ϫ mice after OGD and reduced ischemic brain injury in mutant mice with a deletion in Bid. It was also found that both Bid and caspase-3 are activated in an ischemic brain and in cultured neurons after OGD.The results generated by using BidϪ͞Ϫ mice demonstrate convincingly that Bid plays a prominent role in acute CNS injury. Phenotypically, these mutant mice do not have any CNS developmental defect (11) or any difference in microscopic or macroscopic CNS morphologies, as compared with WT mice. It can be concluded that Bid promotes death in neurons after OGD in vitro and in the brain after f...
Nucleophosmin (NPM)-1 is a multifunctional protein involved in a variety of biologic processes and has been implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments in NPM1 activities, we dissected the C-terminal domain (CTD) into its helical fragments. In this study, we observed the unexpected structural behavior of the peptide fragment corresponding to helix (H)2 (residues 264-277). This peptide has a strong tendency to form amyloidlike assemblies endowed with fibrillar morphology and β-sheet structure, under physiologic conditions, as shown by circular dichroism, thioflavin T, and Congo red binding assays; dynamic light scattering; and atomic force microscopy. The aggregates are also toxic to neuroblastoma cells, as determined using 3-(4;5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and Ca(2+) influx assays. We also found that the extension of the H2 sequence beyond its N terminus, comprising the connecting loop with H1, delayed aggregation and its associated cytotoxicity, suggesting that contiguous regions of H2 have a protective role in preventing aggregation. Our findings and those in the literature suggest that the helical structures present in the CTD are important in preventing harmful aggregation. These findings could elucidate the pathogenesis of acute myeloid leukemia (AML) caused by NPM1 mutants. Because the CTD is not properly folded in these mutants, we hypothesize that the aggregation propensity of this NPM1 region is involved in the pathogenesis of AML. Preliminary assays on NPM1-Cter-MutA, the most frequent AML-CTD mutation, revealed its significant propensity for aggregation. Thus, the aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology.
The EphA2 receptor plays key roles in many physiological and pathological events including cancer. The process of receptor endocytosis and the consequent degradation have lately attracted attention as possible means of overcoming the negative outcomes of EphA2 in cancer cells and decreasing tumor malignancy. A recent study indicates that Sam (Sterile Alpha Motif) domains of Odin, a member of the ANKS (Ankyrin repeat and sterile alpha motif domain-containing) family of proteins, are important to regulate EphA2 endocytosis. Odin contains two tandem Sam domains (Odin-Sam1 and Sam2). Herein we report on the NMR solution structure of Odin-Sam1; through a variety of assays (employing NMR, SPR and ITC techniques), we clearly demonstrate that Odin-Sam1 binds to the Sam domain of EphA2 in the low micromolar range. NMR chemical shift perturbation experiments and molecular modeling studies point out that the two Sam domains interact with a head to tail topology characteristic of several Sam-Sam complexes. This binding mode is similar to that we have previously proposed for the association between the Sam domains of the lipid phosphatase Ship2 and EphA2. This work further validates structural elements relevant for the heterotypic Sam-Sam interactions of EphA2 and provides novel insights for the design of potential therapeutic compounds that can modulate receptor endocytosis.
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