Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of α-synuclein

Sasakawa, Hiroaki; Sakata, Eri; Yamaguchi, Yoshiki; Masuda, M.; Mori, Tetsuya; Kurimoto, Eiji; Iguchi, Takeshi; Hisanaga, Shin‐ichi; Iwatsubo, Takeshi; Hasegawa, Masato; Kato, Koichi

doi:10.1016/j.bbrc.2007.09.048

Cited by 37 publications

(37 citation statements)

References 29 publications

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“…In addition, weak chemical shift changes were observed for the 60 N-terminal residues (Fig. 2C) in S87A ␣-syn, in agreement with recent studies (23 ) couplings were mostly close to their random coil values and were very similar for unphosphorylated WT and phosphorylated Ser-87␣-syn (data not shown), indicating that phosphorylation has no apparent effect on the secondary structure of ␣-syn. Far UV-CD spectra of unphosphorylated WT, S129E, S129D, S129A, and S87A ␣-syn and phosphorylated WT and S87A ␣-syn were virtually identical and consistent with a predominantly random coil structure (Figs.…”

Section: Generation Of Ser(p)-129 and The Phosphomimics S129e/d-supporting

confidence: 77%

Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

Paleologou

Schmid

Rospigliosi

et al. 2008

Journal of Biological Chemistry

335

409

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␣-Synuclein (␣-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related ␣-synulceinopathies. However, whether phosphorylation promotes or inhibits ␣-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of ␣-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type ␣-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated ␣-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of ␣-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of ␣-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern ␣-syn aggregation and toxicity in PD and related ␣-synulceinopathies.

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Section: Generation Of Ser(p)-129 and The Phosphomimics S129e/d-supporting

confidence: 77%

Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

Paleologou

Schmid

Rospigliosi

et al. 2008

Journal of Biological Chemistry

335

409

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“…We have recorded the 1 H, 15 N HSQC spectrum of ␣Syn or ␣Syn(H50Q). The backbone amides of ␣Syn were assigned by using two-dimensional 1 H, 15 N HSQC, three-dimensional HNCACB, CBCA(CO)NH, and NOE experiments together with published data (34). Nearly all of the non-proline backbone amides of ␣Syn in the HSQC experiment were assigned except Asp-2.…”

Section: Resultsmentioning

confidence: 99%

“…Backbone amide assignments of wild type ␣Syn were completed by using two-dimensional 1 H, 15 N HSQC and three-dimensional HNCACB, CBCA-(CO)NH, and NOE experiments together with previously published data (34). Chemical shift changes (⌬␦) were processed by using the formula…”

Section: Methodsmentioning

confidence: 99%

Residue Histidine 50 Plays a Key Role in Protecting α-Synuclein from Aggregation at Physiological pH

Chi

Armstrong

Jones

et al. 2014

Journal of Biological Chemistry

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Background: Mutations of ␣-synuclein (␣Syn) can cause early-onset familial Parkinson disease (PD). Results: The H50Q, H50D, or H50A substitution promotes, whereas the H50R substitution inhibits, ␣Syn aggregation in vitro. Conclusion:The recently identified PD-causing ␣Syn mutant, ␣Syn(H50Q), accelerates ␣Syn aggregation. Significance: The partial positive charge of His-50 at physiological pH likely plays a role in suppressing ␣Syn aggregation.

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“…Recent NMR studies of a-synuclein have shown that phosphorylation at Ser-129 by casein kinase II increases the disordered character of the protein. It was further shown that this structural alteration increased the protein's tendency to selfassociate (Sasakawa et al, 2007). As seen in Figure 8, the structure of Cor47 is left unaltered upon phosphorylation and also shows no tendency to aggregate.…”

Section: Phosphorylation Does Not Generate Any Significant Structuralmentioning

confidence: 88%

Mimicking the Plant Cell Interior under Water Stress by Macromolecular Crowding: Disordered Dehydrin Proteins Are Highly Resistant to Structural Collapse

2008

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The dehydrins are a class of drought-induced proteins in plants that lack a fixed three-dimensional structure. Their specific molecular action, as well as the reason for their disordered character, is as yet poorly understood. It has been speculated, however, that the dehydrins are tuned to acquire a biologically active structure only under the conditions in which they normally function (i.e. upon dehydration). To test this hypothesis, we here investigate the effect of reduced water content and macromolecular crowding on three dehydrins from Arabidopsis (Arabidopsis thaliana). As a simplistic model for mimicking cellular dehydration, we used polyethylene glycol, glycerol, and sugars that plants naturally employ as compatible solutes (i.e. sucrose and glucose). Macromolecular crowding was induced by the large polysaccharides Ficoll and dextran. The results show that the dehydrins are remarkably stable in their disordered state and are only modestly affected by the solvent alterations. A notable exception is the dehydrin Cor47, which shows a small, intrinsic increase in helical structure at high concentrations of osmolytes. We also examined the effect of phosphorylation but found no evidence that such posttranslational modifications of the dehydrin sequences modulate their structural response to osmolytes and crowding agents. These results suggest that the dehydrins are highly specialized proteins that have evolved to maintain their disordered character under conditions in which unfolded states of several globular proteins would tend to collapse.

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Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of α-synuclein

Cited by 37 publications

References 29 publications

Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

Residue Histidine 50 Plays a Key Role in Protecting α-Synuclein from Aggregation at Physiological pH

Mimicking the Plant Cell Interior under Water Stress by Macromolecular Crowding: Disordered Dehydrin Proteins Are Highly Resistant to Structural Collapse

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