title: Semisynthesis and Enzymatic Preparation of Post-translationally Modified α-Synuclein.
abstract: Posttranslational modifications (PTMs) serve as molecular switches for regulating protein folding, function, and interactome and have been implicated in the misfolding and amyloid formation by several proteins linked to neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Understanding the role of individual PTMs in protein misfolding and aggregation requires the preparation of site-specifically modified proteins, as well as the identification of the enzymes involved in regulating these PTMs. Recently, our group has pioneered the development of enzymatic, synthetic, and semisynthetic strategies that allow site-specific introduction of PTMs at single or multiple sites and generation of modified proteins in milligram quantities. In this chapter, we provide detailed description of enzymatic and semisynthetic strategies for the generation of the phosphorylated α-Synuclein (α-Syn) at S129, (pS129), which has been identified as a pathological hallmark of Parkinson's disease. The semisynthetic method described for generation of α-Syn-pS129 requires expertise with protein chemical ligation, but can be used to incorporate other PTMs (single or multiple) within the α-Syn C-terminus if desired. On the other hand, the in vitro kinase-mediated phosphorylation strategy does not require any special setup and is rather easy to apply, but its application is restricted to the generation of α-Syn_pS129. These methods have the potential to increase the availability of pure and homogenous modified α-Syn reagents, which may be used as standards in numerous applications, including the search for potential biomarkers of synucleinopathies.
authors.affiliation: Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, AI 2.151, Station 19, 1015, Lausanne, Switzerland.
authors.forename: Bruno
authors.initials: B
authors.lastname: Fauvet
authors.affiliation: Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, AI 2.151, Station 19, 1015, Lausanne, Switzerland. hilal.lashuel@epfl.ch.|Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar. hilal.lashuel@epfl.ch.
authors.forename: Hilal A
authors.initials: HA
authors.lastname: Lashuel
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D019636:Neurodegenerative Diseases / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D051844:alpha-Synuclein
keywords: Alpha-synuclein; Amyloid; Desulfurization; Native chemical ligation; Parkinson’s disease; Phosphorylation; Posttranslation modification; Semisynthesis