High Speed AFM and NanoInfrared Spectroscopy Investigation of Aß1-42 Peptide Variants and Their Interaction With POPC/SM/Chol/GM1 Model Membranes.

Due to an aging population, neurodegenerative diseases such as Alzheimer's disease (AD) have become a major health issue. In the case of AD, Aß1 - 42 peptides have been identified as one of the markers of the disease with the formation of senile plaques via their aggregation, and could play a role in memory impairment and other tragic syndromes associated with the disease. Many studies have shown that not only the morphology and structure of Aß1 - 42 peptide assembly are playing an important role in the formation of amyloid plaques, but also the interactions between Aß1 - 42 and the cellular membrane are crucial regarding the aggregation processes and toxicity of the amyloid peptides. Despite the increasing amount of information on AD associated amyloids and their toxicity, the molecular mechanisms involved still remain unclear and require in-depth investigation at the local scale to clearly decipher the role of the sequence of the amyloid peptides, of their secondary structures, of their oligomeric states, and of their interactions with lipid membranes. In this original study, through the use of Atomic Force Microscopy (AFM) related-techniques, high-speed AFM and nanoInfrared AFM, we tried to unravel at the nanoscale the link between aggregation state, structure and interaction with membranes in the amyloid/membrane interaction. Using three mutants of Aß peptides, L34T, oG37C, and WT Aß1 - 42 peptides, with differences in morphology, structure and assembly process, as well as model lipidic membranes whose composition and structure allow interactions with the peptides, our AFM study coupling high spatial and temporal resolution and nanoscale structure information clearly evidences a local correlation between the secondary structure of the peptides, their fibrillization kinetics and their interactions with model membranes. Membrane disruption is associated to small transient oligomeric entities in the early stages of aggregation that strongly interact with the membrane, and present an antiparallel ß-sheet secondary structure. The strong effect on membrane integrity that exists when these oligomeric Aß1 - 42 peptides interact with membranes of a particular composition could be a lead for therapeutic studies.

Nanoparticles With a Specific Size and Surface Charge Promote Disruption of the Secondary Structure and Amyloid-Like Fibrillation of Human Insulin Under Physiological Conditions.

Nanoparticles attract much interest as fluorescent labels for diagnostic and therapeutic tools, although their applications are often hindered by size- and shape-dependent cytotoxicity. This cytotoxicity is related not only to the leak of toxic metals from nanoparticles into a biological solution, but also to molecular cytotoxicity effects determined by the formation of a protein corona, appearance of an altered protein conformation leading to exposure of cryptic epitopes and cooperative effects involved in the interaction of proteins and peptides with nanoparticles. In the last case, nanoparticles may serve, depending on their nature, as centers of self-association or fibrillation of proteins and peptides, provoking amyloid-like proteinopathies, or as inhibitors of self-association of proteins, or they can self-assemble on biopolymers as on templates. In this study, human insulin protein was used to analyze nanoparticle-induced proteinopathy in physiological conditions. It is known that human insulin may form amyloid fibers, but only under extreme experimental conditions (very low pH and high temperatures). Here, we have shown that the quantum dots (QDs) may induce amyloid-like fibrillation of human insulin under physiological conditions through a complex process strongly dependent on the size and surface charge of QDs. The insulin molecular structure and fibril morphology have been shown to be modified at different stages of its fibrillation, which has been proved by comparative analysis of the data obtained using circular dichroism, dynamic light scattering, amyloid-specific thioflavin T (ThT) assay, transmission electron microscopy, and high-speed atomic force microscopy. We have found important roles of the QD size and surface charge in the destabilization of the insulin structure and the subsequent fibrillation. Remodeling of the insulin secondary structure accompanied by remarkable increase in the rate of formation of amyloid-like fibrils under physiologically normal conditions was observed when the protein was incubated with QDs of exact specific diameter coated with slightly negative specific polyethylene glycol (PEG) derivatives. Strongly negatively or slightly positively charged PEG-modified QDs of the same specific diameter or QDs of bigger or smaller diameters had no effect on insulin fibrillation. The observed effects pave the way to the control of amyloidosis proteinopathy by varying the nanoparticle size and surface charge.

Real Time and Quantitative Imaging of Lignocellulosic Films Hydrolysis by Atomic Force Microscopy Reveals Lignin Recalcitrance at Nanoscale.

Lignocellulosic biomass is considered as a sustainable source of energy and chemicals, but its recalcitrance to bioconversion still limits its use. In this paper, a strategy based on two aspects was developed to improve our knowledge on the lignin recalcitrance to enzymatic hydrolysis. First, lignocellulosic films of cellulose nanofibrils (CNFs) with increasing content of lignin (up to 40%) were prepared. Thanks to in situ real time Atomic Force Microscopy (AFM) measurements during the hydrolysis and by comparison with biochemical assays, the use of such films allows to fully assess the importance of the lignin content and of the arrangement between CNFs and lignin on the hydrolysis efficiency. In a second time, contrary to other studies by AFM which only followed a specific structure during enzymatic processes mostly on simple systems (CNFs or cellulose nanocrystals), a quantitative analysis of in-situ time-lapse measurements was developed. It enables to accurately address lignocellulosic biomass recalcitrance mechanisms mediated by lignin at nanoscale. Such analysis could pave the way for the use of a quantitative criteria to visualize in situ deconstruction of complex lignocellulosic substrates. Coupling the use of lignocellulosic films and dynamical AFM quantitative analysis to follow the evolution of the structure at nanoscale might lead to an effective targeting of new promising bioconversion strategies.

Conformation-dependent binding of a Tetrastatin peptide to αvß3 integrin decreases melanoma progression through FAK/PI3K/Akt pathway inhibition.

Tetrastatin, a 230 amino acid sequence from collagen IV, was previously demonstrated to inhibit melanoma progression. In the present paper, we identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin in vivo and in vitro on melanoma cell proliferation, migration and invasion. We demonstrated that QS-13 binds to SK-MEL-28 melanoma cells through the αvß3 integrin using blocking antibody and ß3 integrin subunit siRNAs strategies. Relevant QS-13 conformations were extracted from molecular dynamics simulations and their interactions with αVß3 integrin were analyzed by docking experiments to determine the binding areas and the QS-13 amino acids crucial for the binding. The in silico results were confirmed by in vitro experiments. Indeed, QS-13 binding to SK-MEL-28 was dependent on the presence of a disulfide-bound as shown by mass spectroscopy and the binding site on αVß3 was located in close vicinity to the RGD binding site. QS-13 binding inhibits the FAK/PI3K/Akt pathway, a transduction pathway that is largely involved in tumor cell proliferation and migration. Taken together, our results demonstrate that the QS-13 peptide binds αvß3 integrin in a conformation-dependent manner and is a potent antitumor agent that could target cancer cells through αVß3.