Characterization of Oligomeric Intermediates in α-Synuclein Fibrillation: FRET Studies of Y125W/Y133F/Y136F α-Synuclein

Kaylor, Joanna J.; Bodner, Nika; Edridge, Shauna; Yamin, Ghiam; Hong, Dong-Pyo; Fink, Anthony L.

doi:10.1016/j.jmb.2005.08.046

Cited by 148 publications

(193 citation statements)

References 37 publications

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“…We note that while Trp fluorescence has been used to report on the amyloid formation process for the disease-related proteins α-synuclein (38,39) and Aβ 1-40 (40), large changes in hλi and I are not always observed and are highly site dependent. Due to the high sensitivity of W423 emission upon aggregation, we propose that the C-terminal repeats may play a key role in RPT fibril assembly; however, we do not preclude the possibility of involvement of other polypeptide regions.…”

Section: Resultsmentioning

confidence: 96%

Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17

Pfefferkorn

McGlinchey²,

Lee³

2010

Proc. Natl. Acad. Sci. U.S.A.

114

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Pmel17 is a functional amyloidogenic protein whose fibrils act as scaffolds for pigment deposition in human skin and eyes. We have used the repeat domain (RPT, residues 315-444), an essential luminal polypeptide region of Pmel17, as a model system to study conformational changes from soluble unstructured monomers to β-sheet-containing fibrils. Specifically, we report on the effects of solution pH (4 → 7) mimicking pH conditions of melanosomes, acidic organelles where Pmel17 fibrils are formed. Local, secondary, and fibril structure were monitored via intrinsic Trp fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, respectively. We find that W423 is a highly sensitive probe of amyloid assembly with spectral features reflecting local conformational and fibril morphological changes. A critical pH regime (5 AE 0.5) was identified for fibril formation suggesting the involvement of at least three carboxylic acids in the structural rearrangement necessary for aggregation. Moreover, we demonstrate that RPT fibril morphology can be transformed directly by changing solution pH. Based on these results, we propose that intramelanosomal pH regulates Pmel17 amyloid formation and its subsequent dissolution in vivo.fibril | fluorescence | melanosome | tryptophan | melanin

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Section: Resultsmentioning

confidence: 96%

Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17

Pfefferkorn

McGlinchey²,

Lee³

2010

Proc. Natl. Acad. Sci. U.S.A.

114

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“…Prior to measurements, protein solutions were filtered and centrifuged for 30 min at 12,000 ϫ g. The protein concentration was determined by the measurement of absorbance at 275 nm using extinction coefficient ⑀ 275 ϭ 5600 cm Ϫ1 M Ϫ1 . The aggregation protocol was adapted from previous studies (29,30). Monomeric ␣-SN solutions (140 M) in 20 mM HEPES, pH 7.4, were incubated in a Thermomixer comfort (Eppendorf) at 37°C and 700 rpm.…”

Section: Methodsmentioning

confidence: 99%

Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity

Ávila

Torres-Bugeau

Barbosa

et al. 2014

Journal of Biological Chemistry

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Background: Although glycosaminoglycan-induced GAPDH prefibrillar species accelerates ␣-synuclein aggregation, its role in toxicity remains unclear. Results: The toxic effect exerted by ␣-synuclein oligomers on cell culture was abolished by GAPDH protofibril, which was identified and structurally characterized. Conclusion: GAPDH protofibrils can efficiently sequester ␣-synuclein toxic oligomers. Significance: GAPDH protofibrils may play an important role in neuronal proteostasis and could open a novel therapeutic strategy for synucleinopathies.

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“…[15][16][17][18] Diverse oligomeric forms of the protein have also been observed, including short, ring-like protofibrils, short chain protofibrils, 19 and other more globular forms with different secondary structure contents. 20 Because some of the PD linked aS mutations accelerate fibril formation (A53T, E46K) [21][22][23][24][25] while others inhibit it (A30P), 19 but all increase oligomer formation, oligomers have been proposed to be the toxic forms of the protein. 19,21,26,27 The formation of amyloid fibrils by aS occurs via a nucleation dependent pathway, 28 with access to the pathway controlled by conformational intermediates.…”

Section: Introductionmentioning

confidence: 99%

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

2009

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Alpha-synuclein (aS) is the primary component of Lewy bodies, the pathological hallmark of Parkinson's Disease. Aggregation of aS is thought to proceed from a primarily disordered state with nascent secondary structure through intermediate conformations to oligomeric forms and finally to mature amyloid fibrils. Low pH conditions lead to conformational changes associated with increased aS fibril formation. Here we characterize these structural and dynamic changes using solution state NMR measurements of secondary chemical shifts, relaxation parameters, residual dipolar couplings, and paramagnetic relaxation enhancement. We find that the neutralization of negatively charged side-chains eliminates electrostatic repulsion in the C-terminal tail of aS and leads to a collapse of this region at low pH. Hydrophobic contacts between the compact C-terminal tail and the NAC (non-amyloid-b component) region are maintained and may lead to the formation of a globular domain. Transient long-range contacts between the C-terminus of the protein and regions N-terminal to the NAC region are also preserved. Thus, the release of long-range contacts does not play a role in the increased aggregation of aS at low pH, which we instead attribute to the increased hydrophobicity of the protein.

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Characterization of Oligomeric Intermediates in α-Synuclein Fibrillation: FRET Studies of Y125W/Y133F/Y136F α-Synuclein

Cited by 148 publications

References 37 publications

Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17

Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17

Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

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