HIV-1 Nef perturbs artificial membranes: investigation of the contribution of the myristoyl anchor.

Nef, an accessory protein from human immunodeficiency virus type 1, is critical for optimal viral replication and pathogenesis. Here, we analyzed the influence of full-length myristoylated and nonmyristoylated Nef on artificial lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By means of cosedimentation assays, we found that neither nonmyristoylated nor myristoylated Nef stably binds to POPC unilamellar vesicles. Time-resolved ellipsometry rather indicates that the proteins perturb the assembly of POPC planar bilayers. This observation was corroborated by fluorescence and scanning force microscopy, suggesting that membrane disordering occurs upon interaction of full-length myristoylated and nonmyristoylated Nef with planar POPC membranes immobilized on SiO(2) surfaces resulting in loss of material from the surface. The membrane perturbations were further investigated by vesicle release experiments, demonstrating that the disordering results in defects through which the fluorophor carboxyfluorescein can pass. From these results, we conclude that Nef is capable of disordering and perturbing lipid membranes and that the myristoyl group is not the decisive determinant for the action of the protein on lipid membranes.

Fluorescent Nanodiamonds are Efficient, Easy-to-Use Cyto-Compatible Vehicles for Monitored Delivery of Non-Coding Regulatory RNAs.

MicroRNAs are short molecules of RNA regulating most cellular processes via the mechanism of RNA interference. Their dysregulation leads to a disease burden, making them important therapeutic targets. For the successful development of a therapeutic device, the uptake of a functionalized carrier by live cells and the sufficient release of effector therapeutic molecules are limiting factors. Here for the first time, the inhibition of oncogenic microRNA-21 in CT-26 colon cancer cells is achieved, using an advanced nanosystem consisting of fluorescent nanodiamond and antisense RNA. Stable nanocomplexes efficiently deliver antisense RNA into cell cytoplasm, encouraging further study of microRNA-21 function in target cells. Engaging the fluorescent nanoparticle enables monitoring of transfection and release of the antisense RNA load into cell cytoplasm. Importantly, the internalized antisense RNA effectively destroys target microRNA-21 in CT-26 cancer cells. The absence of oncogenic microRNA-21 liberates tumor suppressor genes Pdcd4 and Timp3 from silencing, and results in a decrease of cell invasion and migration, and in the induction of apoptotic cell death. This study uses a nanodiamond-based imaging and delivery system, and shows that the multidimensional performance of the presented device makes nanodiamond-based complexes promising therapeutic devices.

Modulation of insulin degrading enzyme activity and liver cell proliferation.

Diabetes mellitus type 2 (T2DM), insulin therapy, and hyperinsulinemia are independent risk factors of liver cancer. Recently, the use of a novel inhibitor of insulin degrading enzyme (IDE) was proposed as a new therapeutic strategy in T2DM. However, IDE inhibition might stimulate liver cell proliferation via increased intracellular insulin concentration. The aim of this study was to characterize effects of inhibition of IDE activity in HepG2 hepatoma cells and to analyze liver specific expression of IDE in subjects with T2DM. HepG2 cells were treated with 10 nM insulin for 24 h with or without inhibition of IDE activity using IDE RNAi, and cell transcriptome and proliferation rate were analyzed. Human liver samples (n = 22) were used for the gene expression profiling by microarrays. In HepG2 cells, IDE knockdown changed expression of genes involved in cell cycle and apoptosis pathways. Proliferation rate was lower in IDE knockdown cells than in controls. Microarray analysis revealed the decrease of hepatic IDE expression in subjects with T2DM accompanied by the downregulation of the p53-dependent genes FAS and CCNG2, but not by the upregulation of proliferation markers MKI67, MCM2 and PCNA. Similar results were found in the liver microarray dataset from GEO Profiles database. In conclusion, IDE expression is decreased in liver of subjects with T2DM which is accompanied by the dysregulation of p53 pathway. Prolonged use of IDE inhibitors for T2DM treatment should be carefully tested in animal studies regarding its potential effect on hepatic tumorigenesis.