Critical micellar concentration determination of pure phospholipids and lipid-raft and their mixtures with cholesterol.

Phospholipids in biological membranes establish a chemical equilibrium between free phospholipids in the aqueous phase (CMC) and self-assembled phospholipids in vesicles, keeping the CMC constant. The CMC is different for each phospholipid, depends on the amount of cholesterol, and, according to the lipid-chaperone hypothesis, controls the interaction between free phospholipids and amyloidogenic proteins (such as amylin, amyloid-ß, and α-synuclein, all of which are, respectively, associated with a different proteinopathy), which governs the formation of a toxic complex between free lipids and proteins that leads to membrane destruction. Here, we provide quantitative measurements of CMCs and bilayer stability of pure phospholipids, lipid rafts, and their mixture with cholesterol by fluorescence methods (using pyrene as a probe) and light scattering techniques (resonance Rayleigh scattering and fixed-angle light scattering) performed on LUVs, as well as AFM to measure LUV dimensions. Also, we test the lipid-chaperone hypothesis on human IAPP interacting with different mixture of POPC cholesterol. Stated the importance of CMC in membrane stability and protein aggregation processes, these results could be a starting point for the development of a quantitative kinetic model for the lipid chaperone hypothesis.

Probing the helical stability in a VEGF-mimetic peptide.

The analysis of the forces governing helix formation and stability in peptides and proteins has attracted considerable interest in order to shed light on folding mechanism. We analyzed the role of hydrophobic interaction, steric hindrance and chain length on i, i + 3 position in QK peptide, a VEGF mimetic helical peptide. We focused on position 10 of QK, occupied by a leucine, as previous studies highlighted the key role of the Leu7-Leu10 interaction in modulating the helix formation and inducing an unusual thermodynamic stability. Leu10 has been replaced by hydrophobic amino acids with different side-chain length, hydrophobicity and steric hindrance. Ten peptides were, hence, synthesized and analyzed combining circular dichroism, calorimetry and NMR spectroscopy. We found that helical content and thermal stability of peptide QK changed when Leu10 was replaced. Interestingly, we observed that the changes in the helical content and thermal stability were not always correlated and they depend on the type of interaction (strength and geometry) that could be established between Leu7 and the residue in position 10.

The Ionophoric Activity of a Pro-Apoptotic VEGF165 Fragment on HUVEC Cells.

Copper plays an important role as a regulator in many pathologies involving the angiogenesis process. In cancerogenesis, tumor progression, and angiogenic diseases, copper homeostasis is altered. Although many details in the pathways involved are still unknown, some copper-specific ligands have been successfully used as therapeutic agents. Copper-binding peptides able to modulate angiogenesis represent a possible way to value new drugs. We previously reported that a fragment (VEGF73-101) of vascular endothelial growth factor (VEGF165), a potent angiogenic, induced an apoptotic effect on human umbilical vein endothelial cells. The aim of this study was to investigate the putative copper ionophoric activity of VEGF73-101, as well as establish a relationship between the structure of the peptide fragment and the cytotoxic activity in the presence of copper(II) ions. Here, we studied the stoichiometry and the conformation of the VEGF73-101/Cu(II) complexes and some of its mutated peptides by electrospray ionization mass spectrometry and circular dichroism spectroscopy. Furthermore, we evaluated the effect of all peptides in the absence and presence of copper ions by cell viability and cytofuorimetric assays. The obtained results suggest that VEGF73-101 could be considered an interesting candidate in the development of new molecules with ionophoric properties as agents in antiangiogenic therapeutic approaches.

The Role of Cholesterol in Driving IAPP-Membrane Interactions.

Our knowledge of the molecular events underlying type 2 diabetes mellitus-a protein conformational disease characterized by the aggregation of islet amyloid polypeptide (IAPP) in pancreatic ß cells-is limited. However, amyloid-mediated membrane damage is known to play a key role in IAPP cytotoxicity, and therefore the effects of lipid composition on modulating IAPP-membrane interactions have been the focus of intense research. In particular, membrane cholesterol content varies with aging and consequently with adverse environmental factors such as diet and lifestyle, but its role in the development of the disease is controversial. In this study, we employ a combination of experimental techniques and in silico molecular simulations to shed light on the role of cholesterol in IAPP aggregation and the related membrane disruption. We show that if anionic POPC/POPS vesicles are used as model membranes, cholesterol has a negligible effect on the kinetics of IAPP fibril growth on the surface of the bilayer. In addition, cholesterol inhibits membrane damage by amyloid-induced poration on membranes, but enhances leakage through fiber growth on the membrane surface. Conversely, if 1:2 DOPC/DPPC raft-like model membranes are used, cholesterol accelerates fiber growth. Next, it enhances pore formation and suppresses fiber growth on the membrane surface, leading to leakage. Our results highlight a twofold effect of cholesterol on the amyloidogenicity of IAPP and help explain its debated role in type 2 diabetes mellitus.

Non-selective ion channel activity of polymorphic human islet amyloid polypeptide (amylin) double channels.

Fundamental understanding of ion channel formation by amyloid peptides, which is strongly linked to cell toxicity, is very critical for (pre)clinical treatment of neurodegenerative diseases. Here, we combine atomistic simulations and experiments to demonstrate a broad range of conformational states of hIAPP double channels in lipid membranes. All individual channels display high selectivity for Cl(-) ions over cations, but the co-existence of polymorphic double channels of different conformations and orientations with different populations determines the non-ionic selectivity nature of the channels, which is different from the typical amyloid-ß channels that exhibit Ca(2+) selective ion-permeable characteristics. This work provides a more complete physicochemical mechanism of amyloid-channel-induced toxicity.

Exploring the therapeutic potential of Aloin: unraveling neuroprotective and anticancer mechanisms, and strategies for enhanced stability and delivery.

We investigate the therapeutic potential of Aloin A and Aloin B, two natural compounds derived from Aloe vera leaves, focusing on their neuroprotective and anticancer properties. The structural differences between these two epimers suggest that they may exhibit distinct pharmacological properties. Our investigations revealed that both epimers are not stable in aqueous solution and tend to degrade rapidly, with their concentration decreasing by over 50% within approximately 12 h. These results underscore the importance of addressing issues such as the need for encapsulation into effective drug delivery systems to enhance stability. ThT fluorescence experiments showed that neither compound was able to inhibit Aß amyloid aggregation, indicating that other mechanisms may be responsible for their neuroprotective effects. Next, an equimolar mixture of Aloin A and Aloin B demonstrated an ability to inhibit proteasome in tube tests, which is suggestive of potential anticancer properties, in accordance with antiproliferative effects observed in neuroblastoma SH-SY5Y and HeLa cell lines. Higher water stability and increased antiproliferative activity were observed by encapsulation in carbon dot nanoparticles, suggesting a promising potential for further in vivo studies.

Cations as switches of amyloid-mediated membrane disruption mechanisms: calcium and IAPP.

Disruption of the integrity of the plasma membrane by amyloidogenic proteins is linked to the pathogenesis of a number of common age-related diseases. Although accumulating evidence suggests that adverse environmental stressors such as unbalanced levels of metal ions may trigger amyloid-mediated membrane damage, many features of the molecular mechanisms underlying these events are unknown. Using human islet amyloid polypeptide (hIAPP, aka amylin), an amyloidogenic peptide associated with ß-cell death in type 2 diabetes, we demonstrate that the presence of Ca(2+) ions inhibits membrane damage occurring immediately after the interaction of freshly dissolved hIAPP with the membrane, but significantly enhances fiber-dependent membrane disruption. In particular, dye leakage, quartz crystal microbalance, atomic force microscopy, and NMR experiments show that Ca(2+) ions promote a shallow membrane insertion of hIAPP, which leads to the removal of lipids from the bilayer through a detergent-like mechanism triggered by fiber growth. Because both types of membrane-damage mechanisms are common to amyloid toxicity by most amyloidogenic proteins, it is likely that unregulated ion homeostasis, amyloid aggregation, and membrane disruption are all parts of a self-perpetuating cycle that fuels amyloid cytotoxicity.

Lipid composition-dependent membrane fragmentation and pore-forming mechanisms of membrane disruption by pexiganan (MSI-78).

The potency and selectivity of many antimicrobial peptides (AMPs) are correlated with their ability to interact with and disrupt the bacterial cell membrane. In vitro experiments using model membranes have been used to determine the mechanism of membrane disruption of AMPs. Because the mechanism of action of an AMP depends on the ability of the model membrane to accurately mimic the cell membrane, it is important to understand the effect of membrane composition. Anionic lipids that are present in the outer membrane of prokaryotes but are less common in eukaryotic membranes are usually thought to be key for the bacterial selectivity of AMPs. We show by fluorescence measurements of peptide-induced membrane permeabilization that the presence of anionic lipids at high concentrations can actually inhibit membrane disruption by the AMP MSI-78 (pexiganan), a representative of a large class of highly cationic AMPs. Paramagnetic quenching studies suggest MSI-78 is in a surface-associated inactive mode in anionic sodium dodecyl sulfate micelles but is in a deeply buried and presumably more active mode in zwitterionic dodecylphosphocholine micelles. Furthermore, a switch in mechanism occurs with lipid composition. Membrane fragmentation with MSI-78 can be observed in mixed vesicles containing both anionic and zwitterionic lipids but not in vesicles composed of a single lipid of either type. These findings suggest membrane affinity and membrane permeabilization are not always correlated, and additional effects that may be more reflective of the actual cellular environment can be seen as the complexity of the model membranes is increased.

Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes?

The activity of antimicrobial peptides has been shown to depend on the composition of the target cell membrane. The bacterial selectivity of most antimicrobial peptides has been attributed to the presence of abundant acidic phospholipids and the absence of cholesterol in bacterial membranes. The high amount of cholesterol present in eukaryotic cell membranes is thought to prevent peptide-induced membrane disruption by increasing the cohesion and stiffness of the lipid bilayer membrane. While the role of cholesterol on an antimicrobial peptide-induced membrane disrupting activity has been reported for simple, homogeneous lipid bilayer systems, it is not well understood for complex, heterogeneous lipid bilayers exhibiting phase separation (or "lipid rafts"). In this study, we show that cholesterol does not inhibit the disruption of raft-containing 1,2-dioleoyl-sn-glycero-3-phosphocholine:1,2-dipalmitoyol-sn-glycero-3-phosphocholine model membranes by four different cationic antimicrobial peptides, MSI-78, MSI-594, MSI-367 and MSI-843 which permeabilize membranes. Conversely, the presence of cholesterol effectively inhibits the disruption of non-raft containing 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyol-sn-glycero-3-phosphocholine lipid bilayers, even for antimicrobial peptides that do not show a clear preference between the ordered gel and disordered liquid-crystalline phases. Our results show that the peptide selectivity is not only dependent on the lipid phase but also on the presence of phase separation in heterogeneous lipid systems.

Aß8-20 Fragment as an Anti-Fibrillogenic and Neuroprotective Agent: Advancing toward Efficient Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is the most common cause of dementia, characterized by a spectrum of symptoms associated with memory loss and cognitive decline with deleterious consequences in everyday life. The lack of specific drugs for the treatment and/or prevention of this pathology makes AD an ever-increasing economic and social emergency. Oligomeric species of amyloid-beta (Aß) are recognized as the primary cause responsible for synaptic dysfunction and neuronal degeneration, playing a crucial role in the onset of the pathology. Several studies have been focusing on the use of small molecules and peptides targeting oligomeric species to prevent Aß aggregation and toxicity. Among them, peptide fragments derived from the primary sequence of Aß have also been used to exploit any eventual recognition abilities toward the full-length Aß parent peptide. Here, we test the Aß8-20 fragment which contains the self-recognizing Lys-Leu-Val-Phe-Phe sequence and lacks Arg 5 and Asp 7 and the main part of the C-terminus, key points involved in the aggregation pathway and stabilization of the fibrillary structure of Aß. In particular, by combining chemical and biological techniques, we show that Aß8-20 does not undergo random coil to ß sheet conformational transition, does not form amyloid fibrils by itself, and is not toxic for neuronal cells. Moreover, we demonstrate that Aß8-20 mainly interacts with the 4-11 region of Aß1-42 and inhibits the formation of toxic oligomeric species and Aß fibrils. Finally, our data show that Aß8-20 protects neuron-like cells from Aß1-42 oligomer toxicity. We propose Aß8-20 as a promising drug candidate for the treatment of AD.

Stretching the chains: the destabilizing impact of Cu2+ and Zn2+ ions on K48-linked diubiquitin.

Ubiquitin signalling and metal homeostasis play key roles in controlling several physiological cellular activities, including protein trafficking and degradation. While some relationships between these two biochemical pathways have started to surface, our knowledge of their interplay remains limited. Here, we employ a variety of techniques, such as circular dichroism, differential scanning calorimetry, pressure perturbation calorimetry, fluorescence emission, SDS-PAGE, and small-angle X-ray scattering (SAXS) to evaluate the impact of Cu2+ and Zn2+ ions on the structure and stability of K48 linked diubiquitin (K48-Ub2), a simple model for polyubiquitin chains. The SAXS analysis results show that the structure of the metal-free protein is similar to that observed when the protein is bound to the E2 conjugating enzyme, lending support to the idea that the structure of unanchored K48-linked ubiquitin chains is sufficient for identification by conjugating enzymes without the need for an induced fit mechanism. Our results indicate that K48-Ub2 can coordinate up to four metal ions with both copper and zinc ions inducing slight changes to the secondary structure of the protein. However, we noted significant distinctions in their impacts on protein stability and overall architecture. Specifically, Cu2+ ions resulted in a destabilization of the protein structure, which facilitated the formation of dimer aggregates. Next, we observed a shift in the conformational dynamics of K48-Ub2 toward less compact and more flexible states upon metal ion binding, with Zn2+ inducing a more significant effect than Cu2+ ions. Our structural modelling study demonstrates that both metal ions induced perturbations in the K48-Ub2 structure, leading to the separation of the two monomers thus inhibiting interactions with E2 enzymes. In conclusion, the findings from this study enhance our comprehension of the mechanisms underlying Ub chains recognition. Moreover, they strengthen the notion that drug discovery initiatives aimed at targeting metal-mediated disruptions in Ub signaling hold great potential for treating a wide range of diseases that stem from abnormal protein accumulation.

Two-step mechanism of membrane disruption by Aß through membrane fragmentation and pore formation.

Disruption of cell membranes by Aß is believed to be one of the key components of Aß toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aß occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aß(1-40), defects form on the membrane that share many of the properties of Aß channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aß(1-40) is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aß and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.

Phosphatidylethanolamine enhances amyloid fiber-dependent membrane fragmentation.

The toxicity of amyloid-forming peptides has been hypothesized to reside in the ability of protein oligomers to interact with and disrupt the cell membrane. Much of the evidence for this hypothesis comes from in vitro experiments using model membranes. However, the accuracy of this approach depends on the ability of the model membrane to accurately mimic the cell membrane. The effect of membrane composition has been overlooked in many studies of amyloid toxicity in model systems. By combining measurements of membrane binding, membrane permeabilization, and fiber formation, we show that lipids with the phosphatidylethanolamine (PE) headgroup strongly modulate the membrane disruption induced by IAPP (islet amyloid polypeptide protein), an amyloidogenic protein involved in type II diabetes. Our results suggest that PE lipids hamper the interaction of prefibrillar IAPP with membranes but enhance the membrane disruption correlated with the growth of fibers on the membrane surface via a detergent-like mechanism. These findings provide insights into the mechanism of membrane disruption induced by IAPP, suggesting a possible role of PE and other amyloids involved in other pathologies.

Interactions of two O-phosphorylresveratrol derivatives with model membranes.

The hydrosoluble resveratrol derivative 3-O-phosphorylresveratrol was shown to be more cytotoxic against DU 145 prostate cancer cells than its analog 4'-O-phosphorylresveratrol. In an attempt to unveil the molecular determinants that lye at the root of their different biological effects, here we investigate the interactions of the two resveratrol derivatives with DMPC model membranes by using DSC, membrane permeation/poration assays and molecular dynamics. The results show that the 3-O-derivative interacts with DMPC membranes and diffuses across them. The 4'-O-derivative lies preferentially onto the surface of membrane. The MD simulations provide a molecular interpretation of the experiments and highlight that, in order to maximize the apolar interactions, the 3-O-derivative is embedded in the lipid hydrophobic region. This topographical position of the 3-O resveratrol analog perturbs the liquid-crystalline order of the lipid bilayer promoting membrane curvature and partial lipid loss from the vesicle. This finding reconciles with the lowering of the enthalpy of the lipid phase transition and the ability of the molecule to diffuse across membranes. The present data contribute to explain the different biological activity of the two molecules and evidence that membrane permeability is a key requirement for effective design of resveratrol derivatives to be used for therapeutic purposes.

Semax, a Synthetic Regulatory Peptide, Affects Copper-Induced Abeta Aggregation and Amyloid Formation in Artificial Membrane Models.

Alzheimer's disease, the most common form of dementia, is characterized by the aggregation of amyloid beta protein (Aß). The aggregation and toxicity of Aß are strongly modulated by metal ions and phospholipidic membranes. In particular, Cu2+ ions play a pivotal role in modulating Aß aggregation. Although in the last decades several natural or synthetic compounds were evaluated as candidate drugs, to date, no treatments are available for the pathology. Multifunctional compounds able to both inhibit fibrillogenesis, and in particular the formation of oligomeric species, and prevent the formation of the Aß:Cu2+ complex are of particular interest. Here we tested the anti-aggregating properties of a heptapeptide, Semax, an ACTH-like peptide, which is known to form a stable complex with Cu2+ ions and has been proven to have neuroprotective and nootropic effects. We demonstrated through a combination of spectrofluorometric, calorimetric, and MTT assays that Semax not only is able to prevent the formation of Aß:Cu2+ complexes but also has anti-aggregating and protective properties especially in the presence of Cu2+. The results suggest that Semax inhibits fiber formation by interfering with the fibrillogenesis of Aß:Cu2+ complexes.

The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH.

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12-18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as ß-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.

Tau/Aß chimera peptides: A Thioflavin-T and MALDI-TOF study of Aß amyloidosis in the presence of Cu(II) or Zn(II) ions and total lipid brain extract (TLBE) vesicles.

Currently, Alzheimer's Disease (AD) is a complex neurodegenerative condition, with limited therapeutic options. Several factors, like Amyloid ß (Aß) aggregation, tau protein hyperphosphorylation, bio-metals dyshomeostasis and oxidative stress contribute to AD pathogenesis. These pathogenic processes might occur in the aqueous phase but also on neuronal membranes. Thus, investigating the connection between Aß and biomembranes, becomes important for unveiling the molecular mechanism underlying Aß amyloidosis as a critical event in AD pathology. In this work, the interaction of two peptides, made up with hybrid sequences from Tau protein 9-16 (EVMEDHAG) or 26-33 (QGGYTMHQ) N-terminal domain and Aß16-20 (KLVFF) hydrophobic region, with full length Aß40 or Aß42 peptides is reported. The studied "chimera" peptides Ac-EVMEDHAGKLVFF-NH2 (τ9-16-KL) and Ac-QGGYTMHQKLVFF-NH2 (τ26-33-KL) are endowed with Aß recognition and metal ion interaction capabilities provided by the tau or Aß sequences, respectively. These peptides were characterized in previous study along with their metal dependent interaction and amyloidogenesis, either in the presence or absence of metal ion and artificial membranes made up with Total Lipid Brain Extract (TLBE) components, (Sciacca et al., 2020). In the present paper, the ability of the two peptides to inhibit Aß aggregation is studied using composite experimental conditions including aqueous solution, the presence of metal ions (Cu or Zn), the presence of lipid vesicles mimicking neuronal membranes as well as the co-presence of metals and TLBE artificial membranes. We used Thioflavine-T (ThT) fluorescence or MALDI-TOF spectrometry analysis of Aß limited proteolysis to respectively monitor the Aß aggregation kinetic or validation of the Aß interacting regions. We demonstrate that τ9-16-KL and τ26-33-KL peptides differently affect Aß aggregation kinetics, with the tau sequence playing a crucial role. The results are discussed in terms of chimera's peptides hydrophobicity and electrostatic driven interactions at the aqueous/membrane interface.

The interplay between lipid and Aß amyloid homeostasis in Alzheimer's Disease: risk factors and therapeutic opportunities.

Alzheimer's Diseases (AD) is characterized by the accumulation of amyloid deposits of Aß peptide in the brain. Besides genetic background, the presence of other diseases and an unhealthy lifestyle are known risk factors for AD development. Albeit accumulating clinical evidence suggests that an impaired lipid metabolism is related to Aß deposition, mechanistic insights on the link between amyloid fibril formation/clearance and aberrant lipid interactions are still unavailable. Recently, many studies have described the key role played by membrane bound Aß assemblies in neurotoxicity. Moreover, it has been suggested that a derangement of the ubiquitin proteasome pathway and autophagy is significantly correlated with toxic Aß aggregation and dysregulation of lipid levels. Thus, studies focusing on the role played by lipids in Aß aggregation and proteostasis could represent a promising area of investigation for the design of valuable treatments. In this review we examine current knowledge concerning the effects of lipids in Aß aggregation and degradation processes, focusing on the therapeutic opportunities that a comprehensive understanding of all biophysical, biochemical, and biological processes involved may disclose.

Tau/Aß chimera peptides: Evaluating the dual function of metal coordination and membrane interaction in one sequence.

Several abnormal events may concur as major risk factors for Alzheimer's disease (AD) pathogenesis. For instance, dysregulation of brain's metal homeostasis and amyloid-mediated membrane damage are established toxic mechanisms causing neuronal death. In this study, we assess the amyloidogenic propensity and membrane-damage effects, either in the presence or in absence of metal ions, of two newly synthesized bifunctional peptides. These were designed to comprise a metal chelating N-terminus region derived from Tau protein namely the Tau9-16 (EVMEDHAG) or Tau26-33 (QGGYTMHQ) sequences, merged with the C-terminal hydrophobic region analogous to the Amyloid beta (Aß) 16-20 aminoacid sequence KLVFF (KL). Comparative circular dichroism or fluorescence experiments were carried out to look at the peptide conformation, fibril formation and membrane affinity of Tau9-16KL and Tau26-33KL peptides. We found that Tau9-16KL and Tau26-33KL perturb the fibrillogenic process of Aß1-40. Furthermore Cu(II) and, to a lower extent, Zn(II) induced conformational changes Tau26-33KL both in water and in membrane-mimicking environment. By contrast, due to a different metal coordination mode we observed for Tau9-16KL an unstructured peptide conformation in all the experimental conditions. Unlike aqueous solution, a certain propensity to form amyloid structures at the lipid membrane interface clearly emerged for both the peptides. However, the two peptides exhibit a different capability to elicit membrane damage depending on the presence or absence of metal ions. Tau9-16KL and Tau26-33KL can be used as peptide-based molecular systems able to interfere with the metal dependent Aß/Tau cross-seeded generation of membrane active amyloid species.

Trehalose Conjugates of Silybin as Prodrugs for Targeting Toxic Aß Aggregates.

Alzheimer's disease (AD) is linked to the abnormal accumulation of amyloid ß peptide (Aß) aggregates in the brain. Silybin B, a natural compound extracted from milk thistle (Silybum marianum), has been shown to significantly inhibit Aß aggregation in vitro and to exert neuroprotective properties in vivo. However, further explorations of silybin B's clinical potential are currently limited by three main factors: (a) poor solubility, (b) instability in blood serum, and (c) only partial knowledge of silybin's mechanism of action. Here, we address these three limitations. We demonstrate that conjugation of a trehalose moiety to silybin significantly increases both water solubility and stability in blood serum without significantly compromising its antiaggregation properties. Furthermore, using a combination of biophysical techniques with different spatial resolution, that is, TEM, ThT fluorescence, CD, and NMR spectroscopy, we profile the interactions of the trehalose conjugate with both Aß monomers and oligomers and evidence that silybin may shield the "toxic" surfaces formed by the N-terminal and central hydrophobic regions of Aß. Finally, comparative analysis with silybin A, a less active diastereoisomer of silybin B, revealed how even subtle differences in chemical structure may entail different effects on amyloid inhibition. The resulting insight on the mechanism of action of silybins as aggregation inhibitors is anticipated to facilitate the future investigation of silybin's therapeutic potential.