Tryptophan Anchored Peptides in Lipid Bilayer Membranes: Control of Peptide Orientation and the Phase Behavior of Cholesterol-Containing Ternary Lipid Mixtures

Model WALP peptides and next generation WALP-derived hydrophobic model peptides were employed to discover principles that govern protein-lipid interactions in biological membranes. Ternary cholesterol-containing lipid mixtures were examined in the presence of WALP peptides of different lengths (acetyl-GWW(LA)nLWWA-ethanolamide, with n between 3 and 8). Deuterium NMR spectra from labeled lipids reveal that WALP peptides may stabilize lipid ordered raft domains and therefore promote lipid phase separation, albeit to a minor extent. The results depend upon whether dioleolyl- or diphytanoyl-phosphatidylcholine is present as the fluid lipid component. Several WALP peptides were modified to remove anchoring Trp residues from one end or the other, thereby generating half-anchored WALP peptides which have aromatic anchor residues on only one side of the bilayer. The longer half-anchored WALP peptides (having 19-21 residues) were found to have small apparent average tilt values in DLPC, DMPC and DOPC lipid bilayer membranes. The results from a combined 15N PISEMA and 2H GALA analysis--with various analytical treatments of the peptide dynamics--confirmed the small average tilt angle for one of these peptides in DMPC bilayer membranes. Shorter half-anchored WALP peptides with a hydrophobic length theoretically capable of spanning only a monolayer leaflet, however, do not adopt well defined membrane orientations and often aggregate. To a bilayer-spanning half-anchored WALP peptide having Trp17 and Trp18, we incorporated Trp or Arg as a third anchor in position 2 or 6. Incorporation of this third anchor increases the peptide tilt. When the third anchor is positioned at residue 6, the transmembrane conformation becomes destabilized. In GWALP23, acetyl-GGALW(LA)6LWLAGA-ethanolamide, we incorporated Pro-12 (replacing Leu-12) within the transmembrane stretch of the peptide, introducing a distortion in the peptide alpha helix. Based upon combined 2H GALA and 15N PISEMA solid-state NMR experiments and analysis, the segments N-terminal and C-terminal to the proline are tilted by 34°-40° and 27°-29° (± 6°), respectively, with respect to the lipid bilayer normal, and the proline-induced kink angle is 20-23°

Topical ocular application of aggrelyte-2A reduces lens stiffness in mice

Presbyopia is the progressive loss of the ability of the lens to focus on nearby objects due to its increased stiffness. It occurs in the mid-40s and continues to worsen until the mid-60s. The age-associated increase in protein cross-linking in the lens leads to protein aggregation and water insolubility, especially in the nuclear region, contributing to lens stiffness. This study reports the development of aggrelyte-2A (methyl S-acetyl-N-(3,3-dimethylbutanoyl) cysteinate, a derivative of our previously reported aggrelyte-2) for reversing the stiffness of aged lenses. Aggrelyte-2A showed minimal toxicity in cultured mouse lens epithelial cells (up to 2000 µM) and human lens epithelial cells (up to 250 µM). Lenses from aged mice (age: 24-25 months) treated with 1 mM aggrelyte-2A for 24 h, and human lenses (age: 47-67 years) treated with 250 µM aggrelyte-2A for 48 h showed 11-14% reductions in stiffness, accompanied by an increase in acetyllysine in lens proteins, and free-thiols in the lens. Topical application of aggrelyte-2A (40 mM, 5 µl twice daily for 4 weeks) on mouse eyes significantly reduced lens stiffness. The topical application showed no toxicity to the lens, cornea, or retina, as revealed by morphological examination, H&E staining, and optical coherence tomography. These data suggest that aggrelyte-2A could be developed as a presbyopia-reversing therapeutic

Promotion of Protein Solubility and Reduction in Stiffness in Human Lenses by Aggrelyte-1: Implications for Reversing Presbyopia

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58–75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44–66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins.</p