Actin-spectrin scaffold supports open fenestrae in liver sinusoidal endothelial cells.

Fenestrae are open transmembrane pores that are a structural hallmark of healthy liver sinusoidal endothelial cells (LSECs). Their key role is the transport of solutes and macromolecular complexes between the sinusoidal lumen and the space of Disse. To date, the biochemical nature of the cytoskeleton elements that surround the fenestrae and sieve plates in LSECs remain largely elusive. Herein, we took advantage of the latest developments in atomic force imaging and super-resolution fluorescence nanoscopy to define the organization of the supramolecular complex(es) that surround the fenestrae. Our data revealed that spectrin, together with actin, lines the inner cell membrane and provided direct structural support to the membrane-bound pores. We conclusively demonstrated that diamide and iodoacetic acid (IAA) affect fenestrae number by destabilizing the LSEC actin-spectrin scaffold. Furthermore, IAA induces rapid and repeatable switching between the open vs closed state of the fenestrae, indicating that the spectrin-actin complex could play an important role in controlling the pore number. Our results suggest that spectrin functions as a key regulator in the structural preservation of the fenestrae, and as such, it might serve as a molecular target for altering transendothelial permeability.

Studies of G-quadruplexes formed within self-assembled DNA mini-circles.

We have developed self-assembled DNA mini-circles that contain a G-quadruplex-forming sequence from the c-Myc oncogene promoter and demonstrate by FRET that the G-quadruplex unfolding kinetics are 10-fold slower than for the simpler 24-mer G-quadruplex that is commonly used for FRET experiments.

Anthracene-terpyridine metal complexes as new G-quadruplex DNA binders.

The formation of quadruple-stranded DNA induced by planar metal complexes has particular interest in the development of novel anticancer drugs. This is especially relevant for the inhibition of telomerase, which plays an essential role in cancer cell immortalization and is overexpressed in ca. 85-90% of cancer cells. Moreover, G-quadruplexes also exist in other locations in the human genome, namely oncogene promoter regions, and it has been hypothesized that they play a regulatory role in gene transcription. Herein we report a series of new anthracene-containing terpyridine ligands and the corresponding Cu(II) and Pt(II) complexes, with different linkers between the anthracenyl moiety and the terpyridine chelating unit. The interaction of these ligands and metal complexes with different topologies of DNA was studied by several biophysical techniques. The Pt(II) and Cu(II) complexes tested showed affinity for quadruplex-forming sequences with a good selectivity over duplex DNA. Importantly, the free ligands do not have significant affinity for any of the DNA sequences used, which shows that the presence of the metal is essential for high affinity (and selectivity). This effect is more evident in the case of the Pt(II) complexes. Moreover, the presence of a longer linker between the chelating terpyridine unit and the anthracene moiety enhances the interaction with G-quadruplex-forming sequences. We further evaluated the ability of the Cu(II) complexes to interact with, and stabilize G-quadruplex containing regions in oncogene promoters via a polymerase stop assay. These studies indicated that the metal complexes are able to induce G-quadruplex formation and stop polymerase activity.

Copper(II) complexes of substituted salicylaldehyde dibenzyl semicarbazones: synthesis, cytotoxicity and interaction with quadruplex DNA.

A series of substituted salicylaldehyde dibenzyl semicarbazones [RC6H3(OH)CH=N-NHCON(CH2Ph)2] and their copper(II) complexes were synthesized and characterized. The chloridocopper(II) complexes of the 4-OH and 5-OH substituted ligands (complexes 9 and 7) show modest affinity and good selectivity (over duplex DNA) for the quadruplex formed from the human telomeric (HTelo) DNA sequence. Substitution of the chlorido ligands of these two complexes with pyridine yielded derivatives (7-py and 9-py) with increased affinity for HTelo. These derivatives also show good selectivity for HTelo over calf-thymus DNA (170- and 211-fold, respectively). The X-ray crystal structures of 9 and 9-py were determined. Molecular docking studies based on these structures show that the complexes stack on the 5'-end of the HTelo quadruplex, with the hydroxyl group forming a hydrogen bond with a guanine residue. Complexes 7, 9, 7-py and 9-py display significant cytotoxicity against MOLT-4 human leukaemia cells. Interestingly, they have low to negligible cytotoxicity against the non-cancerous IMR-90 human fibroblasts.