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Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: ?-synuclein phosphorylation at tyrosine 125.

ABSTRACT: Despite increasing evidence that supports the role of different post-translational modifications (PTMs) in modulating ?-synuclein (?-syn) aggregation and toxicity, relatively little is known about the functional consequences of each modification and whether or not these modifications are regulated by each other. This lack of knowledge arises primarily from the current lack of tools and methodologies for the site-specific introduction of PTMs in ?-syn. More specifically, the kinases that mediate selective and efficient phosphorylation of C-terminal tyrosine residues of ?-syn remain to be identified. Unlike phospho-serine and phospho-threonine residues, which in some cases can be mimicked by serine/threonine ? glutamate or aspartate substitutions, there are no natural amino acids that can mimic phospho-tyrosine. To address these challenges, we developed a general and efficient semisynthetic strategy that enables the site-specific introduction of single or multiple PTMs and the preparation of homogeneously C-terminal modified forms of ?-syn in milligram quantities. These advances have allowed us to investigate, for the first time, the effects of selective phosphorylation at Y125 on the structure, aggregation, membrane binding, and subcellular localization of ?-syn. The development of semisynthetic methods for the site-specific introduction of single or PTMs represents an important advance toward determining the roles of such modifications in ?-syn structure, aggregation, and functions in heath and disease.

SUBMITTER: Hejjaoui M

PROVIDER: S-EPMC3592575 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: α-synuclein phosphorylation at tyrosine 125.

Hejjaoui Mirva M Butterfield Sara S Fauvet Bruno B Vercruysse Filip F Cui Jia J Dikiy Igor I Prudent Michel M Olschewski Diana D Zhang Yan Y Eliezer David D Lashuel Hilal A HA

Journal of the American Chemical Society 20120313 11

Despite increasing evidence that supports the role of different post-translational modifications (PTMs) in modulating α-synuclein (α-syn) aggregation and toxicity, relatively little is known about the functional consequences of each modification and whether or not these modifications are regulated by each other. This lack of knowledge arises primarily from the current lack of tools and methodologies for the site-specific introduction of PTMs in α-syn. More specifically, the kinases that mediate ...[more]

PMID: 22339654

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Project description:Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by αSyn di-tyrosine dimers. Our findings uncover a complex interplay between S129 phosphorylation and C-terminal tyrosine modifications of αSyn that likely participates in PD pathology.

Dataset Information

Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: ?-synuclein phosphorylation at tyrosine 125.

Publications

Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: α-synuclein phosphorylation at tyrosine 125.

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