Concentration-dependent and surface-assisted self-assembly properties of a bioactive estrogen receptor α-derived peptide

Habchi

Cerreta

et al. 2016

CPD

Self Cite

BackgroundA wide class of human diseases and neurodegenerative disorders, such as Alzheimer’s disease, is due to the failure of a specific peptide or protein to keep its native functional conformational state and to undergo a conformational change into a misfolded state, triggering the formation of fibrillar cross-β sheet amyloid aggregates. During the fibrillization, several coexisting species are formed, giving rise to a highly heterogeneous mixture. Despite its fundamental role in biological function and malfunction, the mechanism of protein self-assembly and the fundamental origins of the connection between aggregation, cellular toxicity and the biochemistry of neurodegeneration remains challenging to elucidate in molecular detail. In particular, the nature of the specific state of proteins that is most prone to cause cytotoxicity is not established.Methods:In the present review, we present the latest advances obtained by Atomic Force Microscopy (AFM) based techniques to unravel the biophysical properties of amyloid aggregates at the nanoscale. Unraveling amyloid single species biophysical properties still represents a formidable experimental challenge, mainly because of their nanoscale dimensions and heterogeneous nature. Bulk techniques, such as circular dichroism or infrared spectroscopy, are not able to characterize the heterogeneity and inner properties of amyloid aggregates at the single species level, preventing a profound investigation of the correlation between the biophysical properties and toxicity of the individual species.Conclusion:The information delivered by AFM based techniques could be central to study the aggregation pathway of proteins and to design molecules that could interfere with amyloid aggregation delaying the onset of misfolding diseases.

Section: Single Molecule Investigation By Atomic Force Microscopy Tecmentioning

confidence: 99%

Section: Single Molecule Investigation By Atomic Force Microscopy Tecmentioning

confidence: 99%

AFM-Based Single Molecule Techniques: Unraveling the Amyloid Pathogenic Species

Habchi

Cerreta

et al. 2016

CPD

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“…2h). This polarization effect demonstrates that the structure of the fibre is anisotropically ordered and that there are more intermolecular β-sheets oriented perpendicular to the fibre axis 22 . We next performed scanning electron microscopy (SEM) to characterize the structural morphology of the FUS phases.…”

Section: Figurementioning

confidence: 90%

“…A silicon gold coated PR-EX-nIR2 (Anasys, USA) cantilever with a nominal radius of 30 nm and an elastic constant of about 0.2 N m -1 was used. To study polarisation effects, the IR light was polarized parallel and perpendicular the surface of deposition 22 . All images were acquired with a resolution of at least 500×500 pixels per line.…”

Section: Methodsmentioning

confidence: 99%

Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition

Shen

Vigolo

et al. 2020

Preprint

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A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1-4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5-7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquidliquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and material properties as a function of shear stress. Our results inform on the molecular grammar underlying protein LST and highlight generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. Taken together, these observations suggest that the shear plays an important role in the irreversible phase transition of liquid-liquid phase separated droplets, shed light on the role of physical factors in driving this transition in protein aggregation related diseases, and open a new route towards artificial shear responsive biomaterials.During silk spinning in nature, the precursor protein molecules stored in liquid-liquid phase separated form undergo a LST to generate fibres mediated by mechanical shear10-13. It is increasingly recognised, however, that a wide range of other proteins can undergo LLPS inside the cell to form condensates14,15.This process is highly dynamic and crucial to cellular function2,5,16, while irreversible phase transitions lead to solid protein aggregates and can result in disease6. Yet, it remains unexplored whether functional protein condensates can also undergo a LST driven by mechanical shear.

“…1c). [24][25][26][27][28][29][30][31][32][33][34] AFM-IR is becoming widely applicable in biology since is capable of acquiring simultaneously morphological, nanomechanical and nanoscale-resolved chemical IR maps and absorption spectra from protein aggregates, liquid-liquid phase separated condensates, chromosomes, and single cells. 29,31,[34][35][36] As fundamental achievement, we have recently demonstrated that AFM-IR enables to acquire infrared absorption spectra for secondary structure determination from single protein molecules.…”

mentioning

confidence: 99%

Infrared Nanospectroscopy Reveals the Molecular Interaction Fingerprint of an Aggregation Inhibitor with Single Aβ42 Oligomers

Habchi

Chia

et al. 2020

Preprint

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Very significant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer's and Parkinson's diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule level morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the interaction between single A42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, originally an anticancer drug capable recently shown to be able to inhibit A42 aggregation in animal models of Alzheimer's disease.Our results demonstrate that the carbonyl group of this compound interacts with A42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as powerful tool in structure-based drug discovery for protein misfolding diseases.Alzheimer's disease (AD) is characterised by memory loss and cognitive impairment. AD is the primary cause of dementia, which affects currently over 50 million people worldwide, a number expected to exceed 150 million by 2050. 1-3 The self-assembly of the 42-residue isoform of the amyloid- (A) peptide into intractable aggregates is considered to be at the core of the molecular pathways leading to AD. [3][4][5] It is therefore important to understand at the molecular level the aggregation process of A42 in order to develop effective therapeutic strategies that aim at inhibiting its self-assembly.Great efforts have been devoted in the last twenty years to understand the molecular basis of this devastating disorder and to develop small molecules that could interfere with the aggregation pathway of A42. 6-12 Indeed, disease-modifying small molecules represent c.a. one third of the registered trials, in which anti-A therapies dominate. 13 However, despite these efforts, no compound has entered the clinical use to date. 13,14 These repeated failures are in part related to an incomplete understanding of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and how they interfere with the pathways of aggregation. It is increasingly recognised that inhibiting A aggregation per se could have unexpected consequences on the toxicity, as it could not only decrease it, but also leave it unaffected, or even increase it. 15 This complexity is due to the non-linear nature of the aggregation network, in which neurotoxic small oligomeric intermediates [16][17][18][19][20] , can be formed in a variety of ways, some of which highly sensitive to small perturbation. Therefore, promising effective therapeutic strategies must be aimed at targeting precise species in a controlled inte...