Quaternary structure of patient-homogenate amplified α-synuclein fibrils modulates seeding of endogenous α-synuclein

Frieg, Benedikt; Geraets, James A.; Strohäker, Timo; Dienemann, Christian; Mavroeidi, Panagiota; Jung, Byung Chul; Kim, Woojin Scott; Lee, Seung-Jae; Xilouri, Maria; Zweckstetter, Markus; Schröder, Gunnar F.

doi:10.1038/s42003-022-03948-y

Cited by 13 publications

(11 citation statements)

References 72 publications

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“…The majority of cryo-EM structures of α-Synuclein fibrils resolved to date showed that at least part of the fibril N-terminus is structured, potentially allowing ThT interactions at those sites 12,[70][71][72] . Also there have been previous reports of ThT-negative α-Synuclein fibrils, suggesting that amyloid aggregation alone is not sufficient for ThT signal 73 .…”

Section: Discussionmentioning

confidence: 99%

An approach to characterize mechanisms of action of anti-amyloidogenic compoundsin vitroandin situ

Stalder,

Serdiuk,

Ghosh

et al. 2023

Preprint

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Aggregation of amyloidogenic proteins is associated with neurodegenerative disease and its modulation is a focus of drug development efforts. However, the physicochemical properties and structural heterogeneity of amyloidogenic proteins hinder the mechanistic understanding of anti- amyloidogenic compounds. Further, modes of interaction with amyloidogenic proteins are often probedin vitrousing purified protein samples, even though these models may not capturein vivoprotein structures and do not enable identification of off-target effects. We have developed a modular structural proteomic pipeline based on limited proteolysis coupled to mass spectrometry (LiP-MS) with improved, amino acid level-resolution, to probe the mechanism of action of anti- amyloidogenic compounds. We demonstrate our approach by analysing the interactions of six known or putative anti-amyloidogenic compounds and the amyloid binder Thioflavin T (ThT) with different structural forms of the amyloidogenic Parkinson’s disease (PD) protein α-Synuclein. Our approach enables determination of putative interaction sites, identification of whether interactions are covalent or non-covalent, and crucially, can probe for interactions of compounds with physiological structures of α-Synuclein in complex cell and tissue extracts and identify off-targets.In vitroanalyses with our pipeline showed that the green tea polyphenol EGCG induces an N- and C-terminus- dependent compaction of the unstructured α-Synuclein monomer, detected preferential interactions of ThT with the N-terminus of α-Synuclein fibrils compared to the amyloid core, and showed that the most potent inhibitors of aggregation in our study (EGCG, baicalein and AC Immune compound #2) induced similar non-fibrillar end structures despite different interactions with α-Synuclein monomers. Importantly, in mammalian cell lysates, α-Synuclein was either a low-affinity target (for EGCG and Baicalein) or did not show evidence of compound interaction (for ThT and doxycycline) in our experimental conditions, despite both monomeric and fibrillar forms interacting with these compoundsin vitro. For EGCG, we validated this result in postmortem brain homogenates from PD patients. Thesein situanalyses identified many additional putative cellular targets of Doxycycline, EGCG, Baicalein and ThT, suggesting that their effects in cellular or animal models of neurodegeneration are likely due to interactions with proteins other than α-Synuclein and showing that anti-amyloidogenic compounds should be analyzedin situas well asin vitro. Our modular pipeline will enablein situscreening of drugs and PET tracers for amyloid aggregates of interest as well as detailed mechanistic studies of compound actionin vitro.

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Section: Discussionmentioning

confidence: 99%

An approach to characterize mechanisms of action of anti-amyloidogenic compoundsin vitroandin situ

Stalder,

Serdiuk,

Ghosh

et al. 2023

Preprint

View full text Add to dashboard Cite

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“…Frieg et al [ 43 ] amplified α-synuclein fibrils from brain extracts of patients with pathologically confirmed PD and multiple system atrophy, determined their 3D structures by Cryo-EM, and evaluated their potential to seed α-synuclein related pathology in oligodendrocytes. In the first step, researchers fibrilized α-synuclein by protein misfolding cyclic amplification (PMCA) (see Section 3 ) using brain extracts of patients.…”

Section: α-Synuclein Misfolding Aggregation and Fibrillationmentioning

confidence: 99%

Synucleins: New Data on Misfolding, Aggregation and Role in Diseases

Surguchov

Surguchev

2022

Biomedicines

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The synucleins are a family of natively unfolded (or intrinsically unstructured) proteins consisting of α-, β-, and γ-synuclein involved in neurodegenerative diseases and cancer. The current number of publications on synucleins has exceeded 16.000. They remain the subject of constant interest for over 35 years. Two reasons explain this unchanging attention: synuclein’s association with several severe human diseases and the lack of understanding of the functional roles under normal physiological conditions. We analyzed recent publications to look at the main trends and developments in synuclein research and discuss possible future directions. Traditional areas of peak research interest which still remain high among last year’s publications are comparative studies of structural features as well as functional research on of three members of the synuclein family. Another popular research topic in the area is a mechanism of α-synuclein accumulation, aggregation, and fibrillation. Exciting fast-growing area of recent research is α-synuclein and epigenetics. We do not present here a broad and comprehensive review of all directions of studies but summarize only the most significant recent findings relevant to these topics and outline potential future directions.

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“…The bends of the peptide backbone chain in region H270 and T275 amino acid residues were obtained. The protein compactization led to a decrease of electrostatic repulsion between monomers for subsequent nucleation and growth phase of the amyloid fibril formation process. − The stability of the PAP(248–286) monomer folded state was shown with the help of molecular dynamics (MD) simulation.…”

Section: Introductionmentioning

confidence: 99%

PAP(248–286) Conformational Changes during the Lag Phase of Amyloid Fibril Formation

2023

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The initial stage of fibril formation of C-terminal region PAP(248–286) of human seminal plasma protein prostatic acid phosphatase was considered. Amyloid fibrils from the peptide PAP(248–286) are termed as a semen-derived enhancer of viral infection (SEVI) found in abundant quantities in semen. The kinetics of the amyloid fibril formation process consists of two characteristic phases (lag phase/nucleation phase and growth phase/elongation phase). The lag phase can be caused by the presence of mature amyloid fibrils (seeds) in protein solution, so-called secondary nucleation. The secondary nucleation includes interaction of protein monomers with the mature fibril surface that leads to protein spatial structural changes for further amyloid fibril formation. In this work, changes of the PAP(248–286) spatial structure were obtained during the secondary nucleation phase. Pulsed-field gradient (PFG) NMR was used to characterize the behavior of monomeric PAP(248–286) in water solution after PAP(248–286) seed addition. The self-diffusion coefficient showed compactization of the peptide monomer due to fibril–monomer interactions. PAP(248–286) spatial structural changes were detected with the help of high-resolution NMR spectroscopy and molecular dynamics (MD) simulation. The folding of PAP(248–286) occurs due to backbone chain bending in the region of H270 and T275 amino acid residues. Obtained folded conformation of PAP(248–286) emerging in the secondary nucleation process is energetically favorable and retains after monomer–amyloid interaction. The structural changes are associated with localization of PAP(248–286) hydrophobic surface regions, which are probably responsible for peptide monomer–amyloid interactions.

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Quaternary structure of patient-homogenate amplified α-synuclein fibrils modulates seeding of endogenous α-synuclein

Cited by 13 publications

References 72 publications

An approach to characterize mechanisms of action of anti-amyloidogenic compoundsin vitroandin situ

An approach to characterize mechanisms of action of anti-amyloidogenic compoundsin vitroandin situ

Synucleins: New Data on Misfolding, Aggregation and Role in Diseases

PAP(248–286) Conformational Changes during the Lag Phase of Amyloid Fibril Formation

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