Semisynthesis and Enzymatic Preparation of Post-translationally Modified α-Synuclein
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.
Key wordsParkinson’s disease Posttranslation modification Alpha-synuclein Amyloid Phosphorylation Semisynthesis Native chemical ligation Desulfurization
This work has been possible thanks to the tremendous efforts of all the members of the Lashuel group that have contributed to developing and optimizing the protocols described here. We wish to specially thank Mr. John Perrin for the enzymatic preparation of α-Syn_pS129; as well as Mr. Anass Chiki, Dr. Sean Deguire, and Dr. John Warner for helpful comments on the manuscript. This work was supported by grants from the European Research Council (ERC grant n° 243182), the Michael J. Fox Foundation for Parkinson’s Research (grant n° 531107), and Ecole Polytechnique Fédérale de Lausanne Swiss National Science Foundation (grant n (31003A_120653).
- 4.Anderson JP, Walker DE, Goldstein JM, de Laat R, Banducci K, Caccavello RJ, Barbour R, Huang J, Kling K, Lee M, Diep L, Keim PS, Shen X, Chataway T, Schlossmacher MG, Seubert P, Schenk D, Sinha S, Gai WP, Chilcote TJ (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J Biol Chem 281:29739–29752CrossRefPubMedGoogle Scholar
- 6.Wang Y, Shi M, Chung KA, Zabetian CP, Leverenz JB, Berg D, Srulijes K, Trojanowski JQ, Lee VM-Y, Siderowf AD, Hurtig H, Litvan I, Schiess MC, Peskind ER, Masuda M, Hasegawa M, Lin X, Pan C, Galasko D, Goldstein DS, Jensen PH, Yang H, Cain KC, Zhang J (2012) Phosphorylated α-Synuclein in Parkinson’s disease. Sci Transl Med 4:121ra120Google Scholar
- 9.Paleologou KE, Schmid AW, Rospigliosi CC, Kim HY, Lamberto GR, Fredenburg RA, Lansbury PT Jr, Fernandez CO, Eliezer D, Zweckstetter M, Lashuel HA (2008) Phosphorylation at Ser-129 but not the phosphomimics S129E/D inhibits the fibrillation of alpha-synuclein. J Biol Chem 283:16895–16905PubMedCentralCrossRefPubMedGoogle Scholar
- 12.Mahul-Mellier AL, Fauvet B, Gysbers A, Dikiy I, Oueslati A, Georgeon S, Lamontanara AJ, Bisquertt A, Eliezer D, Masliah E, Halliday G, Hantschel O, Lashuel HA (2014) c-Abl phosphorylates alpha-synuclein and regulates its degradation: implication for alpha-synuclein clearance and contribution to the pathogenesis of Parkinson’s disease. Hum Mol Genet 23(11):2858–2879PubMedCentralCrossRefPubMedGoogle Scholar
- 19.Fauvet B, Fares MB, Samuel F, Dikiy I, Tandon A, Eliezer D, Lashuel HA (2012) Characterization of semisynthetic and naturally Nalpha-acetylated alpha-synuclein in vitro and in intact cells: implications for aggregation and cellular properties of alpha-synuclein. J Biol Chem 287:28243–28262PubMedCentralCrossRefPubMedGoogle Scholar
- 22.Haj-Yahya M, Fauvet B, Herman-Bachinsky Y, Hejjaoui M, Bavikar SN, Karthikeyan SV, Ciechanover A, Lashuel HA, Brik A (2013) Synthetic polyubiquitinated alpha-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology. Proc Natl Acad Sci U S A 110:17726–17731PubMedCentralCrossRefPubMedGoogle Scholar
- 23.Hejjaoui M, Butterfield SM, Fauvet B, Vercruysse F, Cui J, Dikiy I, Prudent M, Olschewski D, Zhang Y, Eliezer D, Lashuel HA (2012) Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: alpha-synuclein phosphorylation at tyrosine 125. J Am Chem Soc 134(11):5196–5210PubMedCentralCrossRefPubMedGoogle Scholar
- 27.Hejjaoui M (2012) Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis - phosphorylation at Tyrosine 125 and monoubiquitination at Lysine 6, EPFLGoogle Scholar
- 30.PD-10 gravity-flow column user manual: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314723116657/litdoc52130800BB_20110830191706.pdf