Elucidating the Role of C-Terminal Post-Translational Modifications Using Protein Semisynthesis Strategies: α-Synuclein Phosphorylation at Tyrosine 125

Abstract

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 selective and efficient phosphorylation of C-terminal tyrosine residues of α-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 phosphor-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 α-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 α-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 α-syn structure, aggregation and functions in heath and disease.

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

“…4A). It is noteworthy that phosphorylation at Y125 on its own does not influence the conformational or aggregation properties of α-Syn significantly (38). Together, our results suggest that the combination of phosphorylation at Y125 and tetraubiquitination renders α-Syn more susceptible to aggregation and supports our hypothesis for cross-talk between these two types of posttranslational modifications.…”

N-terminal Acetylation Stabilizes N-terminal Helicity in Lipid- and Micelle-bound α-Synuclein and Increases Its Affinity for Physiological Membranes

“…Concentrations of BOG and lipid vesicles were determined using the intensity of alkyl chain peaks in onedimensional proton NMR spectra. R 2 relaxation rates were measured through a series of 9 HSQC-based T 2 experiments with relaxation time delays of 10,10,10,30,30,50,70,90, and 110 ms. The intensity decay of each cross-peak was fit to a single exponential decay function using internal NMRViewJ fitting module.…”

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

“…For example, it is not possible to enzymatically prepare ␣-syn that is site-specifically and homogeneously phosphorylated at Y125 or S87 because the kinases that phosphorylate at these residues also phosphorylate at other sites. For example, Syk is an efficient tyrosine kinase, but it phosphorylates all C-terminal tyrosine residues (57), and the kinases that are specific (such as Lyn, Fyn, and Src) have been found to be inefficient (57,58). Similarly, kinases that efficiently phosphorylate ␣-syn only at S87 remain to be identified; CKI phosphorylates ␣-syn at both S87 and S129 (16,56).…”