Structural differences between toxic and nontoxic HypF-N oligomers

Capitini, Claudia; Patel, Jigar; Natalello, Antonino; D’Andrea, Cristiano; Relini, Annalisa; Jarvis, James A.; Birolo, Leila; Peduzzo, Alessia; Vendruscolo, Michele; Matteini, Paolo; Simone, Alfonso De

doi:10.1039/c8cc03446j

Cited by 26 publications

(79 citation statements)

References 16 publications

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“…31 A 5-μl drop of RT-QuIC product was deposited onto a gold mirror support (ME1S-M01; Thorlabs, Inc., Newton, NJ), followed by air drying for 20 minutes and acquisition of Raman spectra from the outer ring of the dried drop. Backscattered light was collected by a 100× microscope objective with 0.9NA, which generates a 1-μm laser beam waist and a laser power at the sample of 40 mW.…”

Section: Raman Spectroscopymentioning

confidence: 99%

“…Samples for Raman analysis were prepared by repeating the standard RT-QuIC protocol without the addition of ThT in order to avoid any signal interference. 31 A 5-μl drop of RT-QuIC product was deposited onto a gold mirror support (ME1S-M01; Thorlabs, Inc., Newton, NJ), followed by air drying for 20 minutes and acquisition of Raman spectra from the outer ring of the dried drop. A fitting procedure provided a semiquantitative estimate of the secondary structure composition.…”

Section: Raman Spectroscopymentioning

confidence: 99%

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Seeding variability of different alpha synuclein strains in synucleinopathies

Candelise

Schmitz

Llorens

et al. 2019

Annals of Neurology

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Objectives: Currently, the exact reasons why different α-synucleinopathies exhibit variable pathologies and phenotypes are still unknown. A potential explanation may be the existence of distinctive α-synuclein conformers or strains. Here, we intend to analyze the seeding activity of dementia with Lewy bodies (DLB) and Parkinson's disease (PD) brain-derived α-synuclein seeds by real-time quaking-induced conversion (RT-QuIC) and to investigate the structure and morphology of the α-synuclein aggregates generated by RT-QuIC. Methods: A misfolded α-synuclein-enriched brain fraction from frontal cortex and substantia nigra pars compacta tissue, isolated by several filtration and centrifugation steps, was subjected to α-synuclein/RT-QuIC analysis. Our study included neuropathologically well-characterized cases with DLB, PD, and controls (Ctrl). Biochemical and morphological analyses of RT-QuIC products were conducted by western blot, dot blot analysis, Raman spectroscopy, atomic force microscopy, and transmission electron microscopy. Results: Independently from the brain region, we observed different seeding kinetics of α-synuclein in the RT-QuIC in patients with DLB compared to PD and Ctrl. Biochemical characterization of the RT-QuIC product indicated the generation of a proteinase K-resistant and fibrillary α-synuclein species in DLB-seeded reactions, whereas PD and control seeds failed in the conversion of wild-type α-synuclein substrate. Interpretation: Structural variances of α-synuclein seeding kinetics and products in DLB and PD indicated, for the first time, the existence of different α-synuclein strains in these groups. Therefore, our study contributes to a better understanding of the clinical heterogeneity among α-synucleinopathies, offers an opportunity for a specific diagnosis, and opens new avenues for the future development of strain-specific therapies. ANN NEUROL 2019;85:691-703 S ynucleinopathies, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB), are a class of neurodegenerative diseases characterized by the presence of insoluble intraneural deposits consisting of fibrillary α-synuclein, 1,2 referred to as Lewy bodies. A PD with dementia (PDD) diagnosis is appropriate when the cognitive symptoms View this article online at wileyonlinelibrary.com.

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Section: Raman Spectroscopymentioning

confidence: 99%

Section: Raman Spectroscopymentioning

confidence: 99%

Seeding variability of different alpha synuclein strains in synucleinopathies

Candelise

Schmitz

Llorens

et al. 2019

Annals of Neurology

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“…3 Examples of oligomeric diversity include fibrillar and non-fibrillar oligomers, 4 as well as toxic and non-toxic species. 5 A number of studies have demonstrated that oligomeric species are more toxic than fibrillar conformations both in vitro 3,6 and in vivo [7][8] . The binding of conformationally dependant antibodies to a range of toxic protein oligomers suggests that toxicity is directly associated with protein conformation, 9 with toxic oligomeric species being rich in β-sheet secondary structure 6,10 and displaying an increase in hydrophobic residue exposure.…”

Section: Introductionmentioning

confidence: 99%

Conformational Evolution of Molecular Signatures during Amyloidogenic Protein Aggregation

Devitt

Rice

Crisford

et al. 2019

ACS Chem. Neurosci.

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Aggregation is a pathological hallmark of proteinopathies such as Alzheimer's disease and results in the deposition of β-sheet-rich amyloidogenic protein aggregates. Such proteinopathies can be classified by the identity of one or more aggregated protein, with recent evidence also suggesting that distinct molecular conformers (strains) of the same protein can be observed in different diseases, as well is in sub-types of the same disease. Therefore, methods for the quantification of pathological changes in protein conformation are central to understanding and treating proteinopathies. In this work the evolution of Raman spectroscopic molecular signatures of three conformationally distinct proteins, Bovine Serum Albumin (α-helical-rich), β2-microglobulin (β-sheetrich) and tau (natively disordered), was assessed during aggregation into oligomers and fibrils. The morphological evolution was tracked using Atomic Force Microscopy and corresponding conformational changes were assessed by their Raman signatures acquired in both wet and dried conditions. A deconvolution model was developed which allowed us to quantify the conformation of the non-regular protein tau, as well as for the oligomeric and fibrillar species of each of the proteins. Principle component analysis of the fingerprint region allowed further identification of the distinguishing spectral features and unsupervised distinction. While an increase in β-sheet is seen on aggregation, crucially, however, each protein also retains a significant proportion of its native monomeric structure after aggregation. Thus, spectral analysis of each aggregated species, oligomeric, as well as fibrillar, for each protein resulted in a unique and quantitative 'conformational fingerprint'. This approach allowed us to provide the first differential detection of both oligomers and fibrils of the three different amyloidogenic proteins, including tau, whose aggregates have never before been interrogated using spontaneous Raman spectroscopy. Quantitative 'conformational fingerprinting' by Raman spectroscopy thus demonstrates its huge potential and utility in understanding proteinopathic disease mechanisms and for providing strain-specific early diagnostic markers and targets for disease-modifying therapies.

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“…Toxic and nontoxic forms of α‐synuclein oligomers were also found to differently expose hydrophobic regions, in particular a N‐terminus that is required to anchor the toxic species to the membrane, and a β‐sheet structure core that inserts into the membrane . Fluorescence data obtained with ANS, site‐directed labeling with different fluorophores and fluorescence resonance energy transfer (FRET) measurements pointed out to a connection between the toxicity of type A HypF‐N oligomers and the higher solvent‐exposure of three main hydrophobic regions, which encompass 18–30, 55–65, and 75–87 residues . Interestingly, the aromatic residues as well as one of the two His residues (His64) of the HypF‐N molecule are wholly located in these regions (Figure S1, Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Nanoscale Discrimination between Toxic and Nontoxic Protein Misfolded Oligomers with Tip‐Enhanced Raman Spectroscopy

D’Andrea

Foti

Cottat

et al. 2018

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Highly toxic protein misfolded oligomers associated with neurological disorders such as Alzheimer's and Parkinson's diseases are nowadays considered primarily responsible for promoting synaptic failure and neuronal death. Unraveling the relationship between structure and neurotoxicity of protein oligomers appears pivotal in understanding the causes of the pathological process, as well as in designing novel diagnostic and therapeutic strategies tuned toward the earliest and presymptomatic stages of the disease. Here, it is benefited from tip-enhanced Raman spectroscopy (TERS) as a surface-sensitive tool with spatial resolution on the nanoscale, to inspect the spatial organization and surface character of individual protein oligomers from two samples formed by the same polypeptide sequence and different toxicity levels. TERS provides direct assignment of specific amino acid residues that are exposed to a large extent on the surface of toxic species and buried in nontoxic oligomers. These residues, thanks to their outward disposition, might represent structural factors driving the pathogenic behavior exhibited by protein misfolded oligomers, including affecting cell membrane integrity and specific signaling pathways in neurodegenerative conditions.

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Structural differences between toxic and nontoxic HypF-N oligomers

Cited by 26 publications

References 16 publications

Seeding variability of different alpha synuclein strains in synucleinopathies

Seeding variability of different alpha synuclein strains in synucleinopathies

Conformational Evolution of Molecular Signatures during Amyloidogenic Protein Aggregation

Nanoscale Discrimination between Toxic and Nontoxic Protein Misfolded Oligomers with Tip‐Enhanced Raman Spectroscopy

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