Guanine-rich human telomeric DNA can adopt secondary structures known as G-quadruplexes, which can be targeted by small molecules to achieve anticancer effects. So far, the structural information on complexes between human telomeric DNA and ligands is limited to the parallel G-quadruplex conformation, despite the high structural polymorphism of human telomeric G-quadruplexes. No structure has been yet resolved for the complex with telomestatin, one of the most promising G-quadruplex-targeting anticancer drug candidates. Here we present the first high-resolution structure of the complex between an intramolecular (3 + 1) human telomeric G-quadruplex and a telomestatin derivative, the macrocyclic hexaoxazole L2H2-6M(2)OTD. This compound is observed to interact with the G-quadruplex through π-stacking and electrostatic interactions. This structural information provides a platform for the design of topology-specific G-quadruplex-targeting compounds and is valuable for the development of new potent anticancer drugs.
Rif1 regulates replication timing and repair of double-strand DNA breaks. Using a chromatin immunoprecipitation-sequencing method, we identified 35 high-affinity Rif1-binding sites in fission yeast chromosomes. Binding sites tended to be located near dormant origins and to contain at least two copies of a conserved motif, CNWWGTGGGGG. Base substitution within these motifs resulted in complete loss of Rif1 binding and in activation of late-firing or dormant origins located up to 50 kb away. We show that Rif1-binding sites adopt G quadruplex-like structures in vitro, in a manner dependent on the conserved sequence and on other G tracts, and that purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds G quadruplex-like structures at selected intergenic regions, thus generating local chromatin structures that may exert long-range suppressive effects on origin firing.
The rational design of ligands targeting human telomeric DNA G-quadruplexes is a complex problem due to the structural polymorphism that these sequences can adopt in physiological conditions. Moreover, the ability of ligands to switch conformational equilibria between different G-quadruplex structures is often overlooked in docking approaches. Here, we demonstrate that three of the most potent G-quadruplex ligands (360A, Phen-DC3, and pyridostatin) induce conformational changes of telomeric DNA G-quadruplexes to an antiparallel structure (as determined by circular dichroism) containing only one specifically coordinated K(+) (as determined by electrospray mass spectrometry) and, hence, presumably only two consecutive G-quartets. Control ligands TrisQ, known to bind preferentially to hybrid than to antiparallel structures, and L2H2-6M(2)OTD, known not to disrupt the hybrid-1 structure, did not show such K(+) removal. Instead, binding of the cyclic oxazole L2H2-6M(2)OTD was accompanied by the uptake of one additional K(+). Also contrasting with telomeric G-quadruplexes, the parallel-stranded Pu24-myc G-quadruplex, to which Phen-DC3 is known to bind by end-stacking, did not undergo cation removal upon ligand binding. Our study therefore evidences that very affine ligands can induce conformational switching of the human telomeric G-quadruplexes to an antiparallel structure and that this conformational change is accompanied by removal of one interquartet cation.
Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with craniosynostosis (CS), one of the most common congenital craniofacial deformities. Here we describe the creation and analysis of transgenic mice overexpressing Nell-1. Nell-1 transgenic animals exhibited CS-like phenotypes that ranged from simple to compound synostoses. Histologically, the osteogenic fronts of abnormally closing/closed sutures in these animals revealed calvarial overgrowth and overlap along with increased osteoblast differentiation and reduced cell proliferation. Furthermore, anomalies were restricted to calvarial bone, despite generalized, non-tissue-specific overexpression of Nell-1. In vitro, Nell-1 overexpression accelerated calvarial osteoblast differentiation and mineralization under normal culture conditions. Moreover, Nell-1 overexpression in osteoblasts was sufficient to promote alkaline phosphatase expression and micronodule formation. Conversely, downregulation of Nell-1 inhibited osteoblast differentiation in vitro. In summary, Nell-1 overexpression induced calvarial overgrowth resulting in premature suture closure in a rodent model. Nell-1, therefore, has a novel role in CS development, perhaps as part of a complex chain of events resulting in premature suture closure. On a cellular level, Nell-1 expression may modulate and be both sufficient and required for osteoblast differentiation
Recent studies of uncertainty in international relations treat nation-states as unitary actors, but it may be more realistic to suppose that uncertainty also arises from domestic decision-making processes. By formalizing Putnam's theory of two-level games, this article attempts to integrate the studies of uncertainty and the “second image.” The distinction between domestic and international asymmetric information turns out to be subtle yet important. When there is complete information and international asymmetric information, domestic constraints may or may not enhance bargaining power; but they do not make successful ratification difficult. On the other hand, when there is incomplete domestic information, successful ratification is not guaranteed although the expected payoffs of the constrained negotiators may be still greater than without domestic constraints.
The anticancer activity of immune checkpoint inhibitors is attracting attention in various clinical sites. Since green tea catechin has cancer-preventive activity in humans, whether green tea catechin supports the role of immune checkpoint inhibitors was studied. We here report that (−)-epigallocatechin gallate (EGCG) inhibited programmed cell death ligand 1 (PD-L1) expression in non–small-cell lung cancer cells, induced by both interferon (IFN)-γ and epidermal growth factor (EGF). The mRNA and protein levels of IFN-γ–induced PD-L1 were reduced 40–80% after pretreatment with EGCG and green tea extract (GTE) in A549 cells, via inhibition of JAK2/STAT1 signaling. Similarly, EGF-induced PD-L1 expression was reduced about 37–50% in EGCG-pretreated Lu99 cells through inhibition of EGF receptor/Akt signaling. Furthermore, 0.3% GTE in drinking water reduced the average number of tumors per mouse from 4.1 ± 0.5 to 2.6 ± 0.4 and the percentage of PD-L1 positive cells from 9.6% to 2.9%, a decrease of 70%, in lung tumors of A/J mice given a single intraperitoneal injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In co-culture experiments using F10-OVA melanoma cells and tumor-specific CD3+ T cells, EGCG reduced PD-L1 mRNA expression about 30% in F10-OVA cells and restored interleukin-2 mRNA expression in tumor-specific CD3+ T cells. The results show that green tea catechin is an immune checkpoint inhibitor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.