York Tomita

Discovery of embelin as a cell-permeable, small-molecular weight inhibitor of XIAP through structure-based computational screening of a traditional herbal medicine three-dimensional structure database.

The X-linked inhibitor of apoptosis (XIAP) is a promising new molecular target for the design of novel anticancer drugs aiming at overcoming apoptosis-resistance of cancer cells to chemotherapeutic agents and radiation therapy. Recent studies demonstrated that the BIR3 domain of XIAP where caspase-9 and Smac proteins bind is an attractive site for designing small-molecule inhibitors of XIAP. Through computational structure-based screening of an in-house traditional herbal medicine three-dimensional structure database of 8221 individual natural products, followed by biochemical testing of selected candidate compounds, we discovered embelin from the Japanese Ardisia herb as a small-molecular weight inhibitor that binds to the XIAP BIR3 domain. We showed that embelin binds to the XIAP BIR3 protein with an affinity similar to that of the natural Smac peptide using a fluorescence polarization-based binding assay. Our NMR analysis further conclusively confirmed that embelin interacts with several crucial residues in the XIAP BIR3 domain with which Smac and caspsase-9 bind. Embelin inhibits cell growth, induces apoptosis, and activates caspase-9 in prostate cancer cells with high levels of XIAP, but has a minimal effect on normal prostate epithelial and fibroblast cells with low levels of XIAP. In stably XIAP-transfected Jurkat cells, embelin effectively overcomes the protective effect of XIAP to apoptosis and enhances the etoposide-induced apoptosis and has a minimal effect in Jurkat cells transfected with vector control. Taken together, our results showed that embelin is a fairly potent, nonpeptidic, cell-permeable, small-molecule inhibitor of XIAP and represents a promising lead compound for designing an entirely new class of anticancer agents that target the BIR3 domain of XIAP.

WT p53, but Not Tumor-derived Mutants, Bind to Bcl2 via the DNA Binding Domain and Induce Mitochondrial Permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 ± 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.

BRCA1 Regulates Acetylation and Ubiquitination of Estrogen Receptor-α

Inherited mutations of the breast cancer susceptibility gene BRCA1 confer a high risk for breast cancer development. The (300)RXKK and (266)KXK motifs have been identified previously as sites for acetylation of the estrogen receptor-alpha (ER-alpha), and (302)K was also found to be a site for BRCA1-mediated mono-ubiquitination of ER-alpha in vitro. Here we show that ER-alpha proteins with single or double lysine mutations of these motifs (including K303R, a cancer-associated mutant) are resistant to inhibition by BRCA1, even though the mutant ER-alpha proteins retain the ability to bind to BRCA1. We also found that BRCA1 overexpression reduced and knockdown increased the level of acetylated wild-type ER-alpha, without changing the total ER-alpha protein level. Increased acetylation of ER-alpha due to BRCA1 small interfering RNA was dependent upon phosphatidylinositol 3-kinase/Akt signaling and on up-regulation of the coactivator p300. In addition, using an in vitro acetylation assay, we found that in vitro-translated wild-type BRCA1 but not a cancer-associated point mutant (C61G) inhibited p300-mediated acetylation of ER-alpha. Furthermore, BRCA1 overexpression increased the levels of mono-ubiquitinated ER-alpha protein, and a BRCA1 mutant that is defective for ubiquitin ligase activity but retains other BRCA1 functions (I26A) did not ubiquitinate ER-alpha or repress its activity in vivo. Finally, ER-alpha proteins with mutations of the (300)RXKK or (266)KXK motifs showed modest or no BRCA1-induced ubiquitination. We propose a model in which BRCA1 represses ER-alpha activity, in part, by regulating the relative degree of acetylation vs. ubiquitination of ER-alpha.

Chronic carbamazepine selectively downregulates cytosolic phospholipase A2 expression and cyclooxygenase activity in rat brain

BACKGROUND Carbamazepine is a mood stabilizer used as monotherapy or as an adjunct to lithium in the treatment of acute mania or the prophylaxis of bipolar disorder. Based on evidence that lithium and valproate, other mood stabilizers, reduce brain arachidonic acid turnover and its conversion via cyclooxygenase to prostaglandin E(2) in rat brain, one possibility is that carbamazepine also targets the arachidonic acid cascade. METHODS To test this hypothesis, carbamazepine was administered to rats by intraperitoneal injection at a daily dose of 25 mg/kg for 30 days. RESULTS Carbamazepine decreased brain phospholipase A(2) activity and cytosolic phospholipase A(2) protein and messenger RNA levels without changing significantly protein and activity levels of calcium-independent phospholipase A(2) or secretory phospholipase A(2). Cyclooxygenase activity was decreased in carbamazepine-treated rats without any change in cyclooxygenase-1 or cyclooxygenase-2 protein levels. Brain prostaglandin E(2) concentration also was reduced. The protein levels of other arachidonic acid metabolizing enzymes, 5-lipoxygenase and cytochrome P450 epoxygenase, were not significantly changed nor was the brain concentration of the 5-lipoxygenase product leukotriene B(4). CONCLUSIONS Carbamazepine downregulates cytosolic phospholipase A(2)-mediated release of arachidonic acid and its subsequent conversion to prostaglandin E(2) by cyclooxygenase. These effects may contribute to its therapeutic actions in bipolar disorder.

Exploration of Backbone Space in Foldamers Containing α- and β-Amino Acid Residues: Developing Protease-Resistant Oligomers that Bind Tightly to the BH3-Recognition Cleft of Bcl-xL

Protein–protein interactions play crucial roles in cell‐signaling events and are often implicated in human disease. Molecules that bind tightly to functional protein‐surface sites and show high stability to degradative enzymes could be valuable pharmacological tools for dissection of cell‐signaling networks and might ultimately lead to therapeutic agents. We recently described oligomers containing both α‐ and β‐amino acid residues that bind tightly to the BH3 recognition site of the anti‐apoptotic protein Bcl‐xL. The oligomers with highest affinity had a nine‐residue N‐terminal segment with a 1:1 α:β residue repeat and a six‐residue C‐terminal segment containing exclusively proteinogenic α‐residues. The N‐terminal portions of such (α/β+α)‐peptides are highly resistant to proteolysis, but the C‐terminal α‐segments are susceptible. This study emerged from efforts to modify the α‐segment in an (α/β+α)‐peptide in a way that would diminish proteolytic degradation but retain high affinity for Bcl‐xL. Some of the oligomers reported here could prove useful in certain biological applications, particularly those for which extended incubation in a biological milieu is required.

Selective Depletion of Mutant p53 by Cancer Chemopreventive Isothiocyanates and Their Structure−Activity Relationships

Isothiocyanates (ITCs) derived from cruciferous vegetables induce apoptosis in cancer cells. We demonstrate that certain naturally occurring ITCs selectively deplete mutant p53 but not the wild-type and do so via a transcription-independent mechanism. Direct p53 binding followed by conformational changes appears to be a mechanism by which mutant p53 is depleted. Structure-activity relationship studies (SARs) using naturally occurring and synthetic ITCs show that depletion is influenced by the ITC side-chain moiety. Furthermore, we show that cells with p53 mutations are more sensitive to cytotoxicity induced by phenethyl isothiocyanate (PEITC) than those with the wild-type protein. 2,2-Diphenylethyl ITC, a synthetic ITC, is one of the most potent depletors of mutant p53 studies and induces apoptosis to the greatest extent in mutant p53 breast cancer cells. Collectively, this study shows that mutant p53 depletion may be an important novel target for cancer chemoprevention and therapy by natural and synthetic ITCs.

Hydrophile scanning as a complement to alanine scanning for exploring and manipulating protein–protein recognition: Application to the Bim BH3 domain

Alanine scanning has been widely employed as a method of identifying side chains that play important roles in protein–protein and protein–peptide interactions. Here we show how an analogous and complementary technique, hydrophile scanning, can provide additional insight on such interactions. Mutation of a wild‐type residue to alanine removes most of the side‐chain atoms, and the effect of this removal is typically interpreted to indicate contribution of the deleted side chain to the stability of the complex. Hydrophile scanning involves systematic mutation of wild‐type residues to a cationic or anionic residue (lysine or glutamic acid, in this case). We find that the results of these mutations provide insights on interactions between polypeptide surfaces that are complementary to the information obtained via alanine scanning. We have applied this technique to a peptide that corresponds to the BH3 domain of the pro‐apoptotic protein Bim. The wild‐type Bim BH3 domain binds strongly to the anti‐apoptotic proteins Bcl‐xL and Mcl‐1. Combining information from the alanine, lysine, and glutamic acid scans has enabled us to identify Bim BH3 domain mutants containing only two or three sequence changes that bind very selectively either to Bcl‐xL or Mcl‐1. Our findings suggest that hydrophile scanning may prove to be a broadly useful tool for revealing sources of protein–protein recognition and for engineering selectivity into natural sequences

Structural determinants of the BRCA1 : estrogen receptor interaction.

Previously, we showed that the BRCA1 protein interacts directly and functionally with estrogen receptor-alpha (ER-α), resulting in the inhibition of estradiol (E2)-stimulated ER-α transcriptional activity. The interaction sites were mapped to the N-terminus of BRCA1 (within amino acids (aa) 1–302) and the ligand-binding domain/activation function-2 (LBD/AF-2) region (within aa 282–420) of ER-α. In this study, we have further characterized the structure/function relationship for the BRCA1 : ER-α interaction. We found that the N-terminal RING domain (aa 20–64) is not required for the BRCA1 : ER-α interaction. We identified two separate contact points for ER-α, one within aa 1–100 and the other within aa 100–200 of BRCA1; and we showed that each of these BRCA1 peptides interacts with BRCA1 in vitro and in vivo. By using different fragments of the BRCA1 N-terminus, we found that aa 67–100 and 101–133 are required for the interaction with ER-α, but that aa 1–67 and 134–302 are dispensible. Previously, we showed that BRCA1 aa 1–302 does not inhibit E2-stimulated ER-α transcriptional activity but does bind to ER-α and acts as a dominant negative inhibitor of the full-length BRCA1 protein. Somewhat surprisingly, we found that BRCA1 aa 1–100 and BRCA1 aa 101–200 (but not aa 201–300) each inhibited ER-α activity, although not as efficiently as full-length BRCA1. Mutations within an HIV Rev-like nuclear export signal that resembles a nuclear receptor corepressor motif (aa 86–95) impaired the ability of both truncated (aa 1–100) and full-length (aa 1–1863) BRCA1 proteins to interact with and/or repress ER-α activity. Based on these findings, a partial BRCA1 : ER-α three-dimensional structure is proposed. The implications of these findings for understanding the BRCA1 : ER-α interaction are discussed.

Identification of the fibroblast growth factor (FGF)-interacting domain in a secreted FGF-binding protein by phage display

Fibroblast growth factor-binding proteins (FGF-BP) are secreted carrier proteins that release fibroblast growth factors (FGFs) from the extracellular matrix storage and thus enhance FGF activity. Here we have mapped the interaction domain between human FGF-BP1 and FGF-2. For this, we generated T7 phage display libraries of N-terminally and C-terminally truncated FGF-BP1 fragments that were then panned against immobilized FGF-2. From this panning, a C-terminal fragment of FGF-BP1 (amino acids 193-234) was identified as the minimum binding domain for FGF. As a recombinant protein, this C-terminal fragment binds to FGF-2 and enhances FGF-2-induced signaling in NIH-3T3 fibroblasts and GM7373 endothelial cells, as well as mitogenesis and chemotaxis of NIH-3T3 cells. The FGF interaction domain in FGF-BP1 is distinct from the heparin-binding domain (amino acids 110-143), and homology modeling supports the notion of a distinct domain in the C terminus that is conserved across different species. This domain also contains conserved positioning of cysteine residues with the Cys-214/Cys-222 positions in the human protein predicted to participate in disulfide bridge formation. Phage display of a C214A mutation of FGF-BP1 reduced binding to FGF-2, indicating the functional significance of this disulfide bond. We concluded that the FGF interaction domain is contained in the C terminus of FGF-BP1.

Altered GABAergic Neurotransmission is Associated with Increased Kainate-Induced Seizure in Prostaglandin-Endoperoxide Synthase-2 Deficient Mice

Excitotoxicity involves over activation of brain excitatory glutamate receptors and has been implicated in neurological, neurodegenerative and neuropsychiatric diseases. Metabolism of arachidonic acid (AA) through the phospholipase A(2) (PLA(2))/prostaglandin-endoperoxide synthase (PTGS) pathway is increased after excitotoxic stimulation. However, the individual roles of the PTGS isoforms in this process are not well established. We assessed the role of the PTGS isoforms in the process of excitotoxicity by exposing mice deficient in either PTGS-1 (PTGS-1(-/-)) or PTGS-2 (PTGS-2(-/-)) to the prototypic excitotoxin, kainic acid (KA). Seizure intensity and neuronal damage were significantly elevated in KA-exposed PTGS-2(-/-), but not in PTGS-1(-/-), mice. The increased susceptibility was not associated with an alteration in KA receptor binding activity or mediated through the CB1 endocannabinoid receptor. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was decreased in the CA1 pyramidal neurons of PTGS-2(-/-) mice, suggesting an alteration of GABAergic function. In wild-type mice, six weeks treatment with the PTGS-2 selective inhibitor celecoxib recapitulated the increased susceptibility to KA-induced excitotoxicity observed in PTGS-2(-/-) mice, further supporting the role of PTGS-2 in the excitotoxic process. The increased susceptibility to KA was also associated with decreased brain levels of PGE(2), a biomarker of PTGS-2 activity. Our results suggest that PTGS-2 activity and its specific products may modulate neuronal excitability by affecting GABAergic neurotransmission. Further, inhibition of PTGS-2, but not PTGS-1, may increase the susceptibility to seizures.