Delivery of DNA and siRNA by novel gemini-like amphiphilic peptides.

We report the design, synthesis, and characterization of a novel type of cationic lipopeptide, gemini-like amphiphilic peptides or 'geminoids'. As an example, the SPKR peptide, inspired by biological nucleic acid binding motifs, was appended with unsaturated (oleoyl/oleyl) alkyl tails. The compound shows remarkable DNA and siRNA delivery, without lysogenic helper lipid, in a variety of cells, with a moderate cytotoxic effect. It aggregates to nanoparticles that combine with DNA to lipoplexes, which undergo a change from lamellar to the more lysogenic hexagonal packing upon lowering the pH. The versatility of the chemical approach allowed us to study peptides related to SPKR, and to establish that the Pro and at least one of the cationic (Lys, Arg) residues are essential for the biological activity.

Cellular uptake of C4b-binding protein is mediated by heparan sulfate proteoglycans and CD91/LDL receptor-related protein.

C4b-binding protein (C4BP) is a protein acting as a complement inhibitor and a carrier protein for anticoagulant protein S. Previously, we reported that the in vivo clearance of C4BP involves CD91, and that a CD91-interactive site overlaps the heparin-binding site within C4BP alpha-chains 26. Here, we investigated the C4BP-CD91 interaction in more detail. Binding of C4BP to CD91 was unaffected by protein S, which associates with C4BP beta-chain. Second, mutagenesis of cationic residues within C4BP alpha-chains impaired CD91 binding, reducing the affinity of triple mutant C4BPalpha/R39Q-R64Q-R66Q by 20-fold (Kd= 10 nM versus 214 nM for wild-type and mutant C4BP, respectively). Accordingly, intracellular degradation of this mutant by CD91-expressing cells was reduced to levels of CD91-deficient cells. Moreover, C4BPalpha/R39Q-R64Q-R66Q displayed a 3-fold prolonged survival compared to normal C4BP in in vivo clearance experiments. Since these residues also contribute to heparin binding, we explored the role of heparin-sulfate proteoglycans (HSPG) in the endocytosis of C4BP. The absence of HSPG was associated with a near complete absence of cell binding and intracellular degradation of C4BP. Apparently, the cellular uptake of C4BP depends on both HSPG and CD91, involving interactions with positively charged residues within C4BP alpha-chain.

LDL-receptor-related protein regulates beta2-integrin-mediated leukocyte adhesion.

Beta2-integrin clustering on activation is a key event in leukocyte adhesion to the endothelium during the inflammatory response. In the search for molecular mechanisms leading to this clustering, we have identified low-density lipoprotein (LDL) receptor-related protein (LRP) as a new partner for beta2-integrins at the leukocyte surface. Immobilized recombinant LRP fragments served as an adhesive surface for blood-derived leukocytes and the U937 cell line. This adhesion was decreased up to 95% in the presence of antibodies against beta2-integrins, pointing to these integrins as potential partners for LRP. Using purified proteins, LRP indeed associated with the alphaMbeta2 complex and the alphaM and alphaL I-domains (K(d, app) approximately 0.5 microM). Immunoprecipitation experiments and confocal microscopy revealed that endogenously expressed LRP and alphaLbeta2 colocalized in monocytes and U937 cells. Furthermore, activation of U937 cells resulted in clustering of alphaLbeta2 and LRP to similar regions at the cell surface, indicating potential cooperation between both proteins. This was confirmed by the lack of alphaLbeta2 clustering in U937 cells treated by antisense oligonucleotides to down-regulate LRP. In addition, the absence of LRP resulted in complete abrogation of beta2-integrin-dependent adhesion to endothelial cells in a perfusion system, demonstrating the presence of a previously unrecognized link between LRP and leukocyte function.

LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms.

LRP (low-density lipoprotein receptor-related protein) is linked to Alzheimer's disease (AD). Here, we report amyloid beta-peptide Abeta40 binds to immobilized LRP clusters II and IV with high affinity (Kd = 0.6-1.2 nM) compared to Abeta42 and mutant Abeta, and LRP-mediated Abeta brain capillary binding, endocytosis, and transcytosis across the mouse blood-brain barrier are substantially reduced by the high beta sheet content in Abeta and deletion of the receptor-associated protein gene. Despite low Abeta production in the brain, transgenic mice expressing low LRP-clearance mutant Abeta develop robust Abeta cerebral accumulations much earlier than Tg-2576 Abeta-overproducing mice. While Abeta does not affect LRP internalization and synthesis, it promotes proteasome-dependent LRP degradation in endothelium at concentrations > 1 microM, consistent with reduced brain capillary LRP levels in Abeta-accumulating transgenic mice, AD, and patients with cerebrovascular beta-amyloidosis. Thus, low-affinity LRP/Abeta interaction and/or Abeta-induced LRP loss at the BBB mediate brain accumulation of neurotoxic Abeta.

Endocytosed transferrin receptors recycle via distinct dynamin and phosphatidylinositol 3-kinase-dependent pathways.

Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1(G273D)-overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin-1(G273D) on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity.