Sharon Unabia

Cholesterol is necessary both for the toxic effect of Aβ peptides on vascular smooth muscle cells and for Aβ binding to vascular smooth muscle cell membranes

Accumulation of beta amyloid (Aβ) in the brain is central to the pathogenesis of Alzheimers disease. Aβ can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Aβ binding to membranes. Aβ peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Aβ peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Aβ peptides and their membrane binding. ‘Ageing’ the Aβ peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Aβ analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Aβ to purified plasma membrane preparations but also reduced Aβ toxicity. The results support the view that Aβ toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol‐lowering drugs may be of therapeutic benefit because it reduces Aβ‐membrane binding.

The β-amyloid protein of Alzheimer’s disease binds to membrane lipids but does not bind to the α7 nicotinic acetylcholine receptor

Accumulation of the amyloid protein (Aβ) in the brain is an important step in the pathogenesis of Alzheimer’s disease. However, the mechanism by which Aβ exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the α7 nicotinic acetylcholine receptor (α7nAChR). In this study, we examined the binding of Aβ1‐42 to endogenous and recombinantly expressed α7nAChRs. Aβ1‐42 did neither inhibit the specific binding of α7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of α7nAChRs expressed in Xenopus oocytes. Similarly, Aβ1‐42 did not compete for α‐bungarotoxin‐binding sites on SH‐SY5Y cells stably expressing α7nAChRs. The effect of the Aβ1‐42 on tau phosphorylation was also examined. Although Aβ1‐42 altered tau phosphorylation in α7nAChR‐transfected SH‐SY5Y cells, the effect of the peptide was unrelated to α7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Aβ bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Aβ may disrupt membrane lipid structure or fluidity. We conclude that the effects of Aβ are unlikely to be mediated by direct binding to the α7nAChR. Instead, we speculate that Aβ may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1.

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.

A Single β-Amino Acid Substitution to Angiotensin II Confers AT 2 Receptor Selectivity and Vascular Function

Novel AT2R ligands were designed by substituting individual &bgr;-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT2R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT1R- or AT2R- transfected HEK-293 cells, only &bgr;-Asp1-Ang II and Ang II fully displaced [125I]-Ang II from AT1R. In contrast, &bgr;-substitutions at each position of Ang II exhibited AT2R affinity, with &bgr;-Tyr4-Ang II and &bgr;-Ile5-Ang II exhibiting ≈1000-fold AT2R selectivity. In mouse aortic rings, &bgr;-Tyr4-Ang II and &bgr;-Ile5-Ang II evoked vasorelaxation that was sensitive to blockade by the AT2R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT1R blockade, &bgr;-Ile5-Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (&bgr;-Ile5-Ang II plus candesartan, −24±4 mm Hg) to a greater extent than candesartan alone (−11±3 mm Hg, n=7, P<0.05), an effect that was abolished by concomitant PD123319 infusion. However, in an identical experimental protocol, &bgr;-Tyr4-Ang II had no influence on BP (n=10), and it was less stable than &bgr;-Ile5-Ang II in plasma stability assays. Thus, this study demonstrated that a single &bgr;-amino acid substitution resulted in a compound that demonstrated both in vitro vasorelaxation and in vivo depressor activity via AT2R. This approach to the design and synthesis of novel AT2R-selective peptidomimetics shows great potential to provide insight into AT2R function.

Synthesis of Stapled β3-Peptides through Ring-Closing Metathesis

The first synthesis of carbon-stapled beta(3)-peptides is reported. The precursor beta(3)-peptides, with O-allyl beta-serines located in an i/i+3 relationship, were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of these new beta(3)-peptides proceeds smoothly either in solution or on an appropriate solid support. All products were generated with high selectivity for the E-isomer.

Effect of acyl chain structure and bilayer phase state on binding and penetration of a supported lipid bilayer by HPA3

The effect of acyl chain structure and bilayer phase state on binding and penetration by the peptide HPA3 was studied using dual polarisation interferometry. This peptide is an analogue of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1) which has been shown to have antimicrobial and cell-penetrating properties. The binding of HPA3 to zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitolyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and negatively charged membranes composed of DMPC and 1,2-dimyristoyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (DMPG) or POPC and 1-palmitolyl-2-oleyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (POPG) was determined using dual polarisation interferometry (DPI). Mass and birefringence were measured in real time, enabling the creation of birefringence–mass plots for detailed analysis of the changes in lipid bilayer order during the peptide-binding process. HPA3 bound to all four lipids and the binding progressed as a single phase for the saturated gel phase bilayers DMPC and DMPC–DMPG. However, the binding process involved two or more phases, with penetration of the unsaturated fluid phase POPC and POPC–POPG bilayers. Structural changes in the saturated bilayer were partially reversible whereas binding to the unsaturated bilayer resulted in irreversible changes in membrane structure. These results demonstrate that more disordered unsaturated bilayers are more susceptible to further disorganisation and have a lower capacity to recover from peptide-induced structural changes than saturated ordered bilayers. In addition, this study further establishes DPI as powerful tool for analysis of multiphase peptide-insertion processes associated with complex structural changes in the liquid-crystalline membrane.

Conformational stability studies of a stapled hexa-β3-peptide library

A library of 14-helical hexa β(3)-peptides was synthesized in order to determine the influence of sequence variation as well as staple size and location on conformational stability. From this study we show that appropriately stapled hexa-β(3)-peptides can allow for a number of variations without significant perturbation of the 14-helix.

β-amino acid substitution to investigate the recognition of angiotensin II (AngII) by angiotensin converting enzyme 2 (ACE2)

In spite of the important role of angiotensin converting enzyme 2 (ACE2) in the cardiovascular system, little is known about the substrate structural requirements of the AngII–ACE2 interaction. Here we investigate how changes in angiotensin II (AngII) structure affect binding and cleavage by ACE2. A series of C3 β‐amino acid AngII analogs were generated and their secondary structure, ACE2 inhibition, and proteolytic stability assessed by circular dichroism (CD), quenched fluorescence substrate (QFS) assay, and LC‐MS analysis, respectively. The β‐amino acid‐substituted AngII analogs showed differences in secondary structure, ACE2 binding and proteolytic stability. In particular, three different subsets of structure‐activity profiles were observed corresponding to substitutions in the N‐terminus, the central region and the C‐terminal region of AngII. The results show that β‐substitution can dramatically alter the structure of AngII and changes in structure correlated with ACE2 inhibition and/or substrate cleavage. β‐amino acid substitution in the N‐terminal region of AngII caused little change in structure or substrate cleavage, while substitution in the central region of AngII lead to increased β‐turn structure and enhanced substrate cleavage. β‐amino acid substitution in the C‐terminal region significantly diminished both secondary structure and proteolytic processing by ACE2. The β‐AngII analogs with enhanced or decreased proteolytic stability have potential application for therapeutic intervention in cardiovascular disease. Copyright

Tethering of the Proximal Region of the Angiotensin II Receptor (AT1A) C-Terminus to the Cell Membrane

The type 1 angiotensin receptor (AT1A) is a 359 amino acid G protein-coupled receptor that mediates the important cardiovascular and homeostatic actions of the peptide hormone angiotensin II (AngII) [1]. The 54 amino acid carboxyl-terminus (Leu305 to Glu359) of the AT1A receptor interacts with, and activates, G proteins and other signaling molecules, indicating a contribution to receptor activation, while the identification of phosphorylation sites and internalisation motifs suggest a key role in receptor regulation [2]. The C-terminus of the type I angiotensin II receptor (AT1A) is thus a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal region of the C-terminus adopt a helical structure in hydrophobic environments [3], which may relate to the capacity of this region to act as an important switch site in vivo. In order to explore this hypothesis, we examined whether this basic-charged, amphipathic α-helical region can interact with lipid components in the cell membrane and thereby modulate local receptor structure.