No abstract

Claudins are the critical transmembrane proteins in tight junctions. Claudin-5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin-claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin-5 mutants transfected into tight junction-free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans-interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild-type-like trans- and cis-interaction; the disjunction type (blocked trans-interaction); the intracellular type (disturbed folding). Combining site-directed mutagenesis, live-cell imaging-, electron microscopy-, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation.

Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.

The protection and maintenance of the genome in human cells is critical. The cell uses signaling pathways that can down regulate the cell cycle when DNA-damage occurs, giving an opportunity for efficient repair before division.1 However, this raises the question of how cells manage to distinguish the telomeres from DNA double-strand breaks. A six-protein complex named "telosome" or "shelterin" (Figure 1) binds to the double-stranded telomeric DNA and single-stranded telomeric G-overhang at the chromosome ends.2,3 The complex shelters telomeres from the DNA-damage response machinery and protects chromosomes from shortening, nonhomologous end-joining, and homology-directed repair. Moreover, the shelterin component POT1 (protection of telomeres 1)4 has been shown to modulate the activity of telomerase,5 the enzyme capable of conferring infinite proliferative capacity on cells by extension of the G-overhang.6 It has been shown that the telomeric G-overhang, when folded into G-quadruplexes, is resistant to extension by telomerase,7 and that synthetic small molecules that stabilize these structures can decrease the enzyme efficiency.8-10 Gomez et al. showed that the potent G-quadruplex binding natural product telomestatin induces apoptosis of cancer cells via a mechanism proposed to involve the uncapping of POT1 from telomeres.11 Herein, we describe a novel synthetic small molecule (compound 1, Figure 1), which exhibits unprecedented G-quadruplex stabilization leading to an alteration of shelterin at the telomeres of human cancer cells.Compound 1 was designed following intensive research on the biology of G-quadruplex nucleic acids.12 The design rationale comprises certain structural features shared by known quadruplex binding small molecules, with particular emphasis on an electron rich aromatic surface, the potential for a flat conformation, and an ability to participate in hydrogen bonding.13 The small molecule is readily accessible in six synthetic steps that are easily scalable and amenable to molecular diversity (see Supporting Information).We first evaluated the potential for 1 to stabilize the telomeric G-quadruplex by FRETmelting experiments.14 Compound 1 stabilized the human telomeric G-quadruplex with a maximum ΔT m of 35 K in 60 mM K + and 44 K in 100 mM Na + at 0.18 and 0.34 μM compound, respectively. In contrast, the ligand-induced double-stranded DNA stabilization was negligible with a ΔT m of 0.5 K in 60 mM K + at 1 μM compound. It is noteworthy that the G-quadruplex melting profile was almost unaffected by the presence of 25 mol equiv of © 2008 American Chemical Society riou@mnhn.fr; sb10031@cam.ac.uk. Supporting Information Available: Experimental details for the synthesis of 1, FRET-melting, direct telomerase extension assay, in vitro POT1 uncapping assay, POT1 and γH2AX in cellulo experiments, growth inhibition assay. This material is available free of charge via the Internet at http://pubs.acs.org. The data recorded for 1 represent the highest induced shifts in melting temperature for the telomeri...

SummaryHigh-grade serous ovarian cancer (HGSOC) remains an unmet medical challenge. Here, we unravel an unanticipated metabolic heterogeneity in HGSOC. By combining proteomic, metabolomic, and bioergenetic analyses, we identify two molecular subgroups, low- and high-OXPHOS. While low-OXPHOS exhibit a glycolytic metabolism, high-OXPHOS HGSOCs rely on oxidative phosphorylation, supported by glutamine and fatty acid oxidation, and show chronic oxidative stress. We identify an important role for the PML-PGC-1α axis in the metabolic features of high-OXPHOS HGSOC. In high-OXPHOS tumors, chronic oxidative stress promotes aggregation of PML-nuclear bodies, resulting in activation of the transcriptional co-activator PGC-1α. Active PGC-1α increases synthesis of electron transport chain complexes, thereby promoting mitochondrial respiration. Importantly, high-OXPHOS HGSOCs exhibit increased response to conventional chemotherapies, in which increased oxidative stress, PML, and potentially ferroptosis play key functions. Collectively, our data establish a stress-mediated PML-PGC-1α-dependent mechanism that promotes OXPHOS metabolism and chemosensitivity in ovarian cancer.

The exact sites, structures, and molecular mechanisms of interaction between junction organizing zona occludence protein 1 (ZO-1) and the tight junction protein occludin or the adherens junction protein ␣-catenin are unknown. Binding studies by surface plasmon resonance spectroscopy and peptide mapping combined with comparative modeling utilizing crystal structures led for the first time to a molecular model revealing the binding of both occludin and ␣-catenin to the same binding site in ZO-1. Our data support a concept that ZO-1 successively associates with ␣-catenin at the adherens junction and occludin at the tight junction. Strong spatial evidence indicates that the occludin C-terminal coiled-coil domain dimerizes and interacts finally as a four-helix bundle with the identified structural motifs in ZO-1. The helix bundle of occludin 406 -521 and ␣-catenin Different junctional complexes such as adherens junctions and, in specialized tissues, tight junctions, gap junctions, and desmosomes connect cells in multicellular organisms. TJ 1 seal the most apical-lateral parts of cells and constitute a diffusion barrier for the paracellular flow of molecules and serve as a fence between the apical and basolateral membrane compartment (1). In contrast, AJ play important roles in cell adhesion, migration, morphogenesis, and proliferation. AJ represent Ca 2ϩ -dependent cell-cell contacts localized basolateral of TJ, where transmembrane proteins of the cadherin family mediate adhesion. Assembly of AJ is a prerequisite for the formation of TJ and desmosomes (2). ␤-and ␣-catenin bind to the cadherin cytoplasmic domain and link the cadherin-catenin complex to the F-actin cytoskeleton. An important scaffolding protein in cell-cell contacts is the zona occludens protein 1. ZO-1 is a membrane associated guanylate kinase homologue protein composed of the following domains: three PDZ (PSD95/Dlg/ZO-1), a SH3, a GuK (3), an actin binding region (4), and a ZU5 (ZO-1 and Unc5-like netrin receptor domain) according SMART (Simple Modular Architecture Research Tool (smart.embl-heidelberg.de) data base (5)). In non-epithelial cells ZO-1 is a major component of AJ, whereas in epithelial cells it is localized at TJ by directly binding to claudins (6). Occludin, one of the transmembrane proteins of TJ, is a multiphosphoprotein involved in regulation of TJ (7). It has four transmembrane domains with two extracellular loops and a cytosolic N and C terminus. A sequence of 244 amino acids in human ZO-1 containing the GuK domain and an acidic region C terminus to GuK binds to the complete intracellular C-terminal tail of chicken occludin (8).ZO-1 also binds to the AJ protein ␣-catenin (9) and to connexins in gap junctions (10), indicating a general scaffolding function of ZO-1 in junctional complexes. ␣-Catenin consists of several four-helix-bundle domains (vinculin homology domains, VH1-3) and binds ␤-catenin via an intermolecular helix bundle mechanisms within the E-cadherin-catenin complex at the intracellular side of AJ, where one helix of ...

Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.

Nucleic acids containing stretches of tandem guanines can fold into four-stranded structures called G-quadruplexes. The existence of such sequences in genomic DNA suggests the occurrence of these motifs in cells, with potential implications in a number of biological processes relevant to cancer. Small molecules have proven to be valuable tools to dissect cell circuitry. Here, we describe a synthetic small molecule derived from an N,N'-bis(2-quinolinyl)pyridine-2,6-dicarboxamide, which is designed to mediate the selective isolation of G-quadruplex nucleic acids. The methodology was successfully applied to a range of DNA and RNA G-quadruplexes in vitro. We demonstrate the general applicability of the method by isolating telomeric DNA-containing G-quadruplex motifs from cells. We show that telomeres are targets for the probe, providing further evidence of the formation of G-quadruplexes in human cells.