A key factor in the development of Type II diabetes is the loss of insulin producing pancreatic beta-cells. The amyloidogenic human Islet Amyloid Polypeptide (hIAPP also known as human amylin) is believed to play a crucial role in this biological process. Previous studies have shown that hIAPP forms small aggregates that kill beta-cells by disrupting the cellular membrane. In this study, we report membrane fragmentation by hIAPP using solid-state NMR experiments on nanotube arrays of anodic aluminum oxide containing aligned phospholipid membranes. In a narrow concentration range of hIAPP, an isotropic (31)P chemical shift signal indicative of the peptide-induced membrane fragmentation was detected. Solid-state NMR results suggest that membrane fragmentation is related to peptide aggregation as the presence of Congo Red, an inhibitor of amyloid formation, prevented membrane fragmentation and the non-amyloidogenic rat-IAPP did not cause membrane fragmentation. The disappearance of membrane fragmentation at higher concentrations of hIAPP suggests an alternate kinetic pathway to fibril formation in which membrane fragmentation is inhibited.

Insulin-like growth factor binding protein-3 (IGFBP-3) belongs to a family of six IGF binding proteins. We previously found that IGFBP-3 exerts its cytotoxic effects on A549 (p53 wild-type) cell survival through a mechanism that depends on hyaluronan-CD44 interactions. To shed light on the mechanism employed, we used CD44-negative normal human lung cells (HFL1), A549, and H1299 (p53-null) lung cancer cells. A synthetic IGFBP-3 peptide ( 215 -KKGfYKKKQcRpSKGRKR-232 ) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP-3 protein, the peptide blocked hyaluronan-CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP-3 protein or its peptide resulted in increased acetylcholinesterase concentration and activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with increased apoptosis and decreased cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP-3 or its peptide blocks hyaluronan-CD44 signaling via a mechanism that depends on both p53 and acetylcholinesterase.Lung cancer is a devastating human disease and among the most common causes of cancer deaths worldwide 1,2 . Of all cases of the disease, non-small cell lung cancer (NSCLC) accounts for approximately 85% 3 .CD44 is a type 1 transmembrane cell-surface glycoprotein with tumor promoting functions in many types of cancer cells 4-7 . It is the main cell surface receptor for hyaluronan (HA) 5-9 . Found on the extracellular side of the cell membrane is the CD44 globular HA-binding domain (HABD) 9,10 shown previously to bind HA as a globular water-soluble protein 11 . CD44 is encoded by a single gene 5,6,12 and many different variant isoforms (CD44v) are generated by alternative splicing that yield different patterns of amino acid insertion into the stalk domain of CD44 with the smallest being the standard CD44 (CD44s) 5,13-15 . Residues 32-123 in the N-terminal domain of CD44, common to both CD44s and CD44v isoforms, contain the HA-binding motif 16 . Assessment of CD44 expression in human lung cancer cell lines 17 , including A549 and H1299 used in this study, showed that the predominant isoform expressed is CD44s 18 . Being a common marker for tumor-initiating cells/cancer stem cells in human carcinomas, CD44 has gained much attention in the cancer literature 14 . HA-CD44 binding is known to modulate numerous downstream signaling cascades, such as the ERK1/2/MAPK and PI3K/Akt pathways, leading to tumor cell proliferation, survival, chemoresistance, and invasiveness 5,7,12,19 .HA is a non-sulfated, anionic glycosaminoglycan 5,16,20,21 polymer composed of the disaccharide sequence (D-glucuronic acid and D-N-acetylglucosamine) without known post-synthetic modification 6,22-24 ...

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an alpha-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. (15)N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. (31)P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.