Linking protein-protein interactions to the diversity of amyloid-like aggregates

Protein aggregation is often studied in the context of neurodegenerative diseases. Deposits of supramolecular aggregates, often appearing as ordered fibrils, are associated with the onset of devastating pathologies as Alzheimer´s and Parkinson´s diseases. Equally important is the impact that the formation of protein aggregates may have in the quality of a protein drug product. The presence of the so-called sub-visible particles (SVP) and visible particles in protein drug product is indeed considered one of the risk factors potentially inducing immune response in patients. Finally yet importantly, protein aggregates, and particularly amyloid fibrils, have unique structural, physico-chemical, mechanical and optical properties, making them appealing bio-inspired materials for several applications. Either one looks at protein aggregation in the context of diseases, drug development or biomaterials, understanding how protein-protein (PPIs) and protein-solvent interactions (PSI) determine aggregation kinetics and the morphology/structures of the final aggregates is a conditio sine qua non for unraveling the molecular mechanisms ruling the self-assembly reaction and for controlling it. In our group, we have reported the possibility for a large group of proteins under specific destabilizing conditions to form a variety of protein aggregates, being it not limited to the formation of amyloid fibrils (Figure 1) [1]. In line with the scope of the conference, I will present our unique approach based on advanced fluorescence microscopy, small angle X-ray scattering and spectroscopy and aimed at identifying the key PPIs and PSIs responsible for such variability in structures and morphologies [2-6]. We use surfactants, salts, alcohols in bulk and microfluidic setups to finely tune the interactions between proteins and, consequently, control the self-assembly process. Our results show that subtle changes in the PPIs and PSI do not only affect the kinetics, but they may also have a dramatic effect on the 3D arrangement, microscopic structures, mechanical properties and stability of the final self-assembled structures. Our findings provide a scenario in which a pool of highly heterogeneous structures can be generated as a result of interconnected aggregation pathways, being this aspect of key relevance especially for protein drug product development and optimization. Please click Additional Files below to see the full abstract

Early Stage Alpha-Synuclein Amyloid Fibrils are Reservoirs of Membrane-Binding Species

Abstract The presence of αSN fibrils indisputably associates with the development of synucleinopathies. However, while certain fibril morphologies have been linked to downstream pathological phenotypes, others appear less harmful, leading to the concept of fibril strains, originally described in relation to prion disease. Indeed, the presence of fibrils does not associate directly with neurotoxicity. Rather, it has been suggested that the toxic compounds are soluble amyloidogenic oligomers, potentially co-existing with fibrils. Here, combining synchrotron radiation circular dichroism, transmission electron microscopy and binding assays on native plasma membrane sheets, we reveal distinct biological and biophysical differences between initial and matured fibrils, transformed within the timespan of few days. Immature fibrils are reservoirs of membrane-binding species, which in response to even gentle experimental changes release into solution in a reversible manner. In contrast, mature fibrils, albeit macroscopically indistinguishable from their less mature counterparts, are structurally robust, shielding the solution from the membrane active soluble species. We thus show that particular biological activity resides transiently with the fibrillating sample, distinct for one, but not the other, spontaneously formed fibril polymorph. These results shed new light on the principles of fibril polymorphism with consequent impact on future design of assays and therapeutic development

Radiation Hardness and Defects Activity in PEA2PbBr4 Single Crystals

Metal halide perovskites (MHPs) are low-temperature processable hybrid semiconductor materials with exceptional performances that are revolutionizing the field of optoelectronic devices. Despite their great potential, commercial deployment is hindered by MHPs lack of stability and durability, mainly attributed to ions migration and chemical interactions with the device electrodes. To address these issues, 2D layered MHPs have been investigated as possible device interlayers or active material substitutes to reduce ion migration and improve stability. Here we consider the 2D perovskite PEA2PbBr4 that was recently discussed as very promising candidate for X-ray direct detection. While the increased resilience of PEA2PbBr4 detectors have already been reported, the physical mechanisms responsible for such improvement compared to the standard "3D" perovskites are not still fully understood. To unravel the charge transport process in PEA2PbBr4 crystals thought to underly the device better performance, we adapted an investigation technique previously used on highly resistive inorganic semiconductors, called photo induced current transient spectroscopy (PICTS). We demonstrate that PICTS can detect three distinct trap states (T1, T2, and T3) with different activation energies, and that the trap states evolution upon X-ray exposure can explain PEA2PbBr4 superior radiation tolerance and reduced aging effects. Overall, our results provide essential insights into the stability and electrical characteristics of 2D perovskites and their potential application as reliable and direct X-ray detectors

Effect of cholesterol on the interaction between amphyphylic peptides and liposomes

With the rise of antibiotic resistance, antimicrobial peptides (AMPs) have been proposed as an alternative novel class of therapeutic agents. They are polycationic, with a net positive charge of more than +2, and they are characterized by amphipathic structures, with both a hydrophobic and a hydrophilic domain. These characteristics allow them to selectively bind to negatively charged lipids (largely present in bacteria, not in mammalian cells), via hydrophobic and electrostatic interactions. Moreover, mammalian cells are characterized by a high content of cholesterol. For this reason, here we present an experimental study on the effect of the presence of cholesterol on the capability of amphyphylic peptide Trasportant 10 (TP10) to interact with model membranes with selected composition. The study was performed by means of fluorescence spectroscopy and fluorescence confocal microscopy measurements also exploiting the advantages of phasor plot analysis of Fluorescence Lifetime Imaging (FLIM) measurements. Our results show that the presence of cholesterol inhibits TP-10 interaction with lipid vesicles, the extent of the observed effect being dependent on the cholesterol concentration in the membrane

Flexibility defines structure in crystals of amphiphilic DNA nanostars.

DNA nanostructures with programmable shape and interactions can be used as building blocks for the self-assembly of crystalline materials with prescribed nanoscale features, holding a vast technological potential. Structural rigidity and bond directionality have been recognised as key design features for DNA motifs to sustain long-range order in 3D, but the practical challenges associated with prescribing building-block geometry with sufficient accuracy have limited the variety of available designs. We have recently introduced a novel platform for the one-pot preparation of crystalline DNA frameworks supported by a combination of Watson-Crick base pairing and hydrophobic forces (Brady et al 2017 Nano Lett. 17 3276-81). Here we use small angle x-ray scattering and coarse-grained molecular simulations to demonstrate that, as opposed to available all-DNA approaches, amphiphilic motifs do not rely on structural rigidity to support long-range order. Instead, the flexibility of amphiphilic DNA building-blocks is a crucial feature for successful crystallisation

Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures.

Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena.This is the author's accepted manuscript and will be under embargo until the 3rd of September 2015. The final version is published by ACS in The Journal of Physical Chemistry Letters here: http://pubs.acs.org/doi/abs/10.1021/jz501614e

Il ruolo del substrato di rame nella sintesi di grafene cresciuto per deposizione chimica da fase vapore

La Chemical Vapor Deposition (CVD) permette la crescita di sottili strati di grafene con aree di decine di centimetri quadrati in maniera continua ed uniforme. Questa tecnica utilizza un substrato metallico, solitamente rame, riscaldato oltre i 1000 °C, sulla cui superficie il carbonio cristallizza sotto forma di grafene in un’atmosfera attiva di metano ed idrogeno. Durante la crescita, sulla superficie del rame si decompone il metano utilizzato come sorgente di carbonio. La morfologia e la composizione della superficie del rame diventano quindi elementi critici del processo per garantire la sintesi di grafene di alta qualità e purezza. In questo manoscritto si documenta l’attività sperimentale svolta presso i laboratori dell’Istituto per la Microelettronica e i Microsistemi del CNR di Bologna sulla caratterizzazione della superficie del substrato di rame utilizzato per la sintesi del grafene per CVD. L’obiettivo di questa attività è stato la caratterizzazione della morfologia superficiale del foglio metallico con misure di rugosità e di dimensione dei grani cristallini, seguendo l’evoluzione di queste caratteristiche durante i passaggi del processo di sintesi. Le misure di rugosità sono state effettuate utilizzando tecniche di profilometria ottica interferometrica, che hanno permesso di misurare l’effetto di livellamento successivo all' introduzione di un etching chimico nel processo consolidato utilizzato presso i laboratori dell’IMM di Bologna. Nell'ultima parte di questo manoscritto si è invece studiato, con tecniche di microscopia ottica ed elettronica a scansione, l’effetto di diverse concentrazioni di argon e idrogeno durante il trattamento termico di annealing del rame sulla riorganizzazione dei suoi grani cristallini. L’analisi preliminare effettuata ha permesso di individuare un intervallo ottimale dei parametri di annealing e di crescita del grafene, suggerendo importanti direzioni per migliorare il processo di sintesi attualmente utilizzato

The route to protein aggregate superstructures:Particulates and amyloid-like spherulites

AbstractDepending on external conditions, native proteins may change their structure and undergo different association routes leading to a large scale polymorphism of the aggregates. This feature has been widely observed but is not fully understood yet. This review focuses on morphologies, physico-chemical properties and mechanisms of formation of amyloid structures and protein superstructures. In particular, the main focus will be on protein particulates and amyloid-like spherulites, briefly summarizing possible experimental methods of analysis. Moreover, we will highlight the role of protein conformational changes and dominant forces in driving association together with their connection with the final aggregate structure. Eventually, we will discuss future perspectives in this field and we will comment what is, in our opinion, urgently needed